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NORMAL GENOMIC

• Lenneman BR, Rothman-Denes LB
• Structural and biochemical investigation of bacteriophage N4-encoded RNA polymerases.
• Biomolecules. 2015; 5: 647-67
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• Bacteriophage N4 regulates the temporal expression of its genome through the activity of three distinct RNA polymerases (RNAP). Expression of the early genes is carried out by a phage-encoded, virion-encapsidated RNAP (vRNAP) that is injected into the host at the onset of infection and transcribes the early genes. These encode the components of new transcriptional machinery (N4 RNAPII and cofactors) responsible for the synthesis of middle RNAs. Both N4 RNAPs belong to the T7-like "single-subunit" family of polymerases. Herein, we describe their mechanisms of promoter recognition, regulation, and roles in the phage life cycle.

• Dangkulwanich M, Ishibashi T, Bintu L, Bustamante C
• Molecular mechanisms of transcription through single-molecule experiments.
• Chem Rev. 2014; 114: 3203-23

• Pandey M, Patel SS
• Helicase and polymerase move together close to the fork junction and copy DNA in one-nucleotide steps.
• Cell Rep. 2014; 6: 1129-38
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• By simultaneously measuring DNA synthesis and dNTP hydrolysis, we show that T7 DNA polymerase and T7 gp4 helicase move in sync during leading-strand synthesis, taking one-nucleotide steps and hydrolyzing one dNTP per base-pair unwound/copied. The cooperative catalysis enables the helicase and polymerase to move at a uniformly fast rate without guanine:cytosine (GC) dependency or idling with futile NTP hydrolysis. We show that the helicase and polymerase are located close to the replication fork junction. This architecture enables the polymerase to use its strand-displacement synthesis to increase the unwinding rate, whereas the helicase aids this process by translocating along single-stranded DNA and trapping the unwound bases. Thus, in contrast to the helicase-only unwinding model, our results suggest a model in which the helicase and polymerase are moving in one-nucleotide steps, DNA synthesis drives fork unwinding, and a role of the helicase is to trap the unwound bases and prevent DNA reannealing.

• Schaerli Y, Gili M, Isalan M
• A split intein T7 RNA polymerase for transcriptional AND-logic.
• Nucleic Acids Res. 2014; 42: 12322-8
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• Synthetic biology has developed numerous parts for building synthetic gene circuits. However, few parts have been described for prokaryotes to integrate two signals at a promoter in an AND fashion, i.e. the promoter is only activated in the presence of both signals. Here we present a new part for this function: a split intein T7 RNA polymerase. We divide T7 RNA polymerase into two expression domains and fuse each to a split intein. Only when both domains are expressed does the split intein mediate protein trans-splicing, yielding a full-length T7 RNA polymerase that can transcribe genes via a T7 promoter. We demonstrate an AND gate with the new part: the signal-to-background ratio is very high, resulting in an almost digital signal. This has utility for more complex circuits and so we construct a band-pass filter in Escherichia coli. The split intein approach should be widely applicable for engineering artificial gene circuit parts.

• Boehr DD
• The ins and outs of viral RNA polymerase translocation.
• J Mol Biol. 2014; 426: 1373-6

• Molodtsov V, Anikin M, McAllister WT
• The presence of an RNA:DNA hybrid that is prone to slippage promotes termination by T7 RNA polymerase.
• J Mol Biol. 2014; 426: 3095-107
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• Intrinsic termination signals for multisubunit bacterial RNA polymerases (RNAPs) encode a GC-rich stem-loop structure followed by a polyuridine [poly(U)] tract, and it has been proposed that steric clash of the stem-loop with the exit pore of the RNAP imposes a shearing force on the RNA in the downstream RNA:DNA hybrid, resulting in misalignment of the active site. The structurally unrelated T7 RNAP terminates at a similar type of signal (TPhi), suggesting a common mechanism for termination. In the absence of a hairpin (passive conditions), T7 RNAP slips efficiently in both homopolymeric A and U tracts, and we have found that replacement of the U tract in TPhi with a slippage-prone A tract still allows efficient termination. Under passive conditions, incorporation of a single G residue following a poly(U) tract (which is the situation during termination at TPhi) results in a "locked" complex that is unable to extend the transcript. Our results support a model in which transmission of the shearing force generated by steric clash of the hairpin with the exit pore is promoted by the presence of a slippery tracts downstream, resulting in alterations in the active site and the formation of a locked complex that represents an early step in the termination pathway.

• Deshpande AP, Sultana S, Patel SS
• Fluorescent methods to study transcription initiation and transition into elongation.
• EXS. 2014; 105: 105-30
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• The DNA-dependent RNA polymerases induce specific conformational changes in the promoter DNA during transcription initiation. Fluorescence spectroscopy sensitively monitors these DNA conformational changes in real time and at equilibrium providing powerful ways to estimate interactions in transcriptional complexes and to assess how transcription is regulated by the promoter DNA sequence, transcription factors, and small ligands. Ensemble fluorescence methods described here probe the individual steps of promoter binding, bending, opening, and transition into the elongation using T7 phage and mitochondrial transcriptional systems as examples.

• Mekler V, Minakhin L, Borukhov S, Mustaev A, Severinov K
• Coupling of downstream RNA polymerase-promoter interactions with formation of catalytically competent transcription initiation complex.
• J Mol Biol. 2014; 426: 3973-84
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• Bacterial RNA polymerase (RNAP) makes extensive contacts with duplex DNA downstream of the transcription bubble in initiation and elongation complexes. We investigated the role of downstream interactions in formation of catalytically competent transcription initiation complex by measuring initiation activity of stable RNAP complexes with model promoter DNA fragments whose downstream ends extend from +3 to +21 relative to the transcription start site at +1. We found that DNA downstream of position +6 does not play a significant role in transcription initiation when RNAP-promoter interactions upstream of the transcription start site are strong and promoter melting region is AT rich. Further shortening of downstream DNA dramatically reduces efficiency of transcription initiation. The boundary of minimal downstream DNA duplex needed for efficient transcription initiation shifted further away from the catalytic center upon increasing the GC content of promoter melting region or in the presence of bacterial stringent response regulators DksA and ppGpp. These results indicate that the strength of RNAP-downstream DNA interactions has to reach a certain threshold to retain the catalytically competent conformation of the initiation complex and that establishment of contacts between RNAP and downstream DNA can be coupled with promoter melting. The data further suggest that RNAP interactions with DNA immediately downstream of the transcription bubble are particularly important for initiation of transcription. We hypothesize that these active center-proximal contacts stabilize the DNA template strand in the active center cleft and/or position the RNAP clamp domain to allow RNA synthesis.

• Toriumi K, Tsukahara T, Hanai R
• R-Loop Formation In Trans at an AGGAG Repeat.
• J Nucleic Acids. 2013; 2013: 629218-629218
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• Formation of RNA-DNA hybrid, or R-loop, was studied in vitro by transcribing an AGGAG repeat with T7 RNA polymerase. When ribonuclease T1 was present, R-loop formation in cis was diminished, indicating that the transcript was separated from the template and reassociated with it. The transcript was found to form an R-loop in trans with DNA comprising the AGGAG repeat, when the DNA was supercoiled. Results of chemical modification indicated that the duplex opened at the AGGAG repeat under negative supercoiling.

• Wang B, Feig M, Cukier RI, Burton ZF
• Computational simulation strategies for analysis of multisubunit RNA polymerases.
• Chem Rev. 2013; 113: 8546-66

• Sun H, Jiang S, Caton-Williams J, Liu H, Huang Z
• 2-Selenouridine triphosphate synthesis and Se-RNA transcription.
• RNA. 2013; 19: 1309-14
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• 2-Selenouridine ((Se)U) is one of the naturally occurring modifications of Se-tRNAs ((Se)U-RNA) at the wobble position of the anticodon loop. Its role in the RNA-RNA interaction, especially during the mRNA decoding, is elusive. To assist the research exploration, herein we report the enzymatic synthesis of the (Se)U-RNA via 2-selenouridine triphosphate ((Se)UTP) synthesis and RNA transcription. Moreover, we have demonstrated that the synthesized (Se)UTP is stable and recognizable by T7 RNA polymerase. Under the optimized conditions, the transcription yield of (Se)U-RNA can reach up to 85% of the corresponding native RNA. Furthermore, the transcribed (Se)U-hammerhead ribozyme has the similar activity as the corresponding native, which suggests usefulness of (Se)U-RNAs in function and structure studies of noncoding RNAs, including the Se-tRNAs.

• Liu B, Lin J, Steitz TA
• Structure of the PolIIIalpha-tauc-DNA complex suggests an atomic model of the replisome.
• Structure. 2013; 21: 658-64
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• The C-terminal domain (CTD) of the tau subunit of the clamp loader (tauc) binds to both the DnaB helicase and the DNA polymerase III alpha subunit (PolIIIalpha), and determines their relative positions and orientations on the leading and lagging strands. Here, we present a 3.2 A resolution structure of Thermus aquaticus PolIIIalpha in complex with tauc and a DNA substrate. The structure reveals that the CTD of tauc interacts with the CTD of PolIIIalpha through its C-terminal helix and the adjacent loop. Additionally, in this complex PolIIIalpha displays an open conformation that includes the reorientations of the oligonucleotide-binding fold and the thumb domain, which may be an indirect result of crystal packing due to the presence of the tauc. Nevertheless, the position of the tauc on PolIIIalpha allows us to suggest an approximate model for how the PolIIIalpha is oriented and positioned on the DnaB helicase.

• Schwinghammer K, Cheung AC, Morozov YI, Agaronyan K, Temiakov D, Cramer P
• Structure of human mitochondrial RNA polymerase elongation complex.
• Nat Struct Mol Biol. 2013; 20: 1298-303
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• Here we report the crystal structure of the human mitochondrial RNA polymerase (mtRNAP) transcription elongation complex, determined at 2.65-A resolution. The structure reveals a 9-bp hybrid formed between the DNA template and the RNA transcript and one turn of DNA both upstream and downstream of the hybrid. Comparisons with the distantly related RNA polymerase (RNAP) from bacteriophage T7 indicates conserved mechanisms for substrate binding and nucleotide incorporation but also strong mechanistic differences. Whereas T7 RNAP refolds during the transition from initiation to elongation, mtRNAP adopts an intermediary conformation that is capable of elongation without refolding. The intercalating hairpin that melts DNA during T7 RNAP initiation separates RNA from DNA during mtRNAP elongation. Newly synthesized RNA exits toward the pentatricopeptide repeat (PPR) domain, a unique feature of mtRNAP with conserved RNA-recognition motifs.

• Weinzierl RO
• The RNA polymerase factory and archaeal transcription.
• Chem Rev. 2013; 113: 8350-76

• Da LT, Pardo Avila F, Wang D, Huang X
• A two-state model for the dynamics of the pyrophosphate ion release in bacterial RNA polymerase.
• PLoS Comput Biol. 2013; 9: 1003020-1003020
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• The dynamics of the PPi release during the transcription elongation of bacterial RNA polymerase and its effects on the Trigger Loop (TL) opening motion are still elusive. Here, we built a Markov State Model (MSM) from extensive all-atom molecular dynamics (MD) simulations to investigate the mechanism of the PPi release. Our MSM has identified a simple two-state mechanism for the PPi release instead of a more complex four-state mechanism observed in RNA polymerase II (Pol II). We observed that the PPi release in bacterial RNA polymerase occurs at sub-microsecond timescale, which is approximately 3-fold faster than that in Pol II. After escaping from the active site, the (Mg-PPi)(2-) group passes through a single elongated metastable region where several positively charged residues on the secondary channel provide favorable interactions. Surprisingly, we found that the PPi release is not coupled with the TL unfolding but correlates tightly with the side-chain rotation of the TL residue R1239. Our work sheds light on the dynamics underlying the transcription elongation of the bacterial RNA polymerase.

• Belostotskii AA
• [Analysis of protein-on-DNA binding profiles, detected with chIP-seq method, reveals possible interaction of specific transcription factors with RNA polymerase II in the process of transcription elongation].
• Biofizika. 2012; 57: 215-20
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• It is thought that in the course of mRNA transcription almost all transcription factors stay on a promoter while RNA polymerase II "clears" the promoter and "proceeds" to elongation. However, analysis of some specific transcription factors and RNA polymerase II binding profiles on DNA, detected with ChIP-seq method, revealed the possibility of interaction between transcription factors and RNA polymerase II in the process of transcription elongation.

• Chakraborty A et al.
• Opening and closing of the bacterial RNA polymerase clamp.
• Science. 2012; 337: 591-5
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• Using single-molecule fluorescence resonance energy transfer, we have defined bacterial RNA polymerase (RNAP) clamp conformation at each step in transcription initiation and elongation. We find that the clamp predominantly is open in free RNAP and early intermediates in transcription initiation but closes upon formation of a catalytically competent transcription initiation complex and remains closed during initial transcription and transcription elongation. We show that four RNAP inhibitors interfere with clamp opening. We propose that clamp opening allows DNA to be loaded into and unwound in the RNAP active-center cleft, that DNA loading and unwinding trigger clamp closure, and that clamp closure accounts for the high stability of initiation complexes and the high stability and processivity of elongation complexes.

• Deshpande AP, Patel SS
• Mechanism of transcription initiation by the yeast mitochondrial RNA polymerase.
• Biochim Biophys Acta. 2012; 1819: 930-8
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• Mitochondria are the major supplier of cellular energy in the form of ATP. Defects in normal ATP production due to dysfunctions in mitochondrial gene expression are responsible for many mitochondrial and aging related disorders. Mitochondria carry their own DNA genome which is transcribed by relatively simple transcriptional machinery consisting of the mitochondrial RNAP (mtRNAP) and one or more transcription factors. The mtRNAPs are remarkably similar in sequence and structure to single-subunit bacteriophage T7 RNAP but they require accessory transcription factors for promoter-specific initiation. Comparison of the mechanisms of T7 RNAP and mtRNAP provides a framework to better understand how mtRNAP and the transcription factors work together to facilitate promoter selection, DNA melting, initiating nucleotide binding, and promoter clearance. This review focuses primarily on the mechanistic characterization of transcription initiation by the yeast Saccharomyces cerevisiae mtRNAP (Rpo41) and its transcription factor (Mtf1) drawing insights from the homologous T7 and the human mitochondrial transcription systems. We discuss regulatory mechanisms of mitochondrial transcription and the idea that the mtRNAP acts as the in vivo ATP "sensor" to regulate gene expression. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

• Minczuk M et al.
• TEFM (c17orf42) is necessary for transcription of human mtDNA.
• Nucleic Acids Res. 2011; 39: 4284-99
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• Here we show that c17orf42, hereafter TEFM (transcription elongation factor of mitochondria), makes a critical contribution to mitochondrial transcription. Inactivation of TEFM in cells by RNA interference results in respiratory incompetence owing to decreased levels of H- and L-strand promoter-distal mitochondrial transcripts. Affinity purification of TEFM from human mitochondria yielded a complex comprising mitochondrial transcripts, mitochondrial RNA polymerase (POLRMT), pentatricopeptide repeat domain 3 protein (PTCD3), and a putative DEAD-box RNA helicase, DHX30. After RNase treatment only POLRMT remained associated with TEFM, and in human cultured cells TEFM formed foci coincident with newly synthesized mitochondrial RNA. Based on deletion mutants, TEFM interacts with the catalytic region of POLRMT, and in vitro TEFM enhanced POLRMT processivity on ss- and dsDNA templates. TEFM contains two HhH motifs and a Ribonuclease H fold, similar to the nuclear transcription elongation regulator Spt6. These findings lead us to propose that TEFM is a mitochondrial transcription elongation factor.

• Bai Y, Srivastava SK, Chang JH, Manley JL, Tong L
• Structural basis for dimerization and activity of human PAPD1, a noncanonical poly(A) polymerase.
• Mol Cell. 2011; 41: 311-20
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• Poly(A) polymerases (PAPs) are found in most living organisms and have important roles in RNA function and metabolism. Here, we report the crystal structure of human PAPD1, a noncanonical PAP that can polyadenylate RNAs in the mitochondria (also known as mtPAP) and oligouridylate histone mRNAs (TUTase1). The overall structure of the palm and fingers domains is similar to that in the canonical PAPs. The active site is located at the interface between the two domains, with a large pocket that can accommodate the substrates. The structure reveals the presence of a previously unrecognized domain in the N-terminal region of PAPD1, with a backbone fold that is similar to that of RNP-type RNA binding domains. This domain (named the RL domain), together with a beta-arm insertion in the palm domain, contributes to dimerization of PAPD1. Surprisingly, our mutagenesis and biochemical studies show that dimerization is required for the catalytic activity of PAPD1.

• Grohmann D, Werner F
• Cycling through transcription with the RNA polymerase F/E (RPB4/7) complex: structure, function and evolution of archaeal RNA polymerase.
• Res Microbiol. 2011; 162: 10-8
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• RNA polymerases (RNAPs) from the three domains of life, Bacteria, Archaea and Eukarya, are evolutionarily related and thus have common structural and functional features. Despite the radically different morphology of Archaea and Eukarya, their RNAP subunit composition and utilisation of basal transcription factors are almost identical. This review focuses on the multiple functions of the most prominent feature that differentiates these enzymes from the bacterial RNAP--a stalk-like protrusion, which consists of the heterodimeric F/E subcomplex. F/E is highly versatile, it facilitates DNA strand-separation during transcription initiation, increases processivity during the elongation phase of transcription and ensures efficient transcription termination.

• Mayer MJ, Garefalaki V, Spoerl R, Narbad A, Meijers R
• Structure-based modification of a Clostridium difficile-targeting endolysin affects activity and host range.
• J Bacteriol. 2011; 193: 5477-86
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• Endolysin CD27L causes cell lysis of the pathogen Clostridium difficile, a major cause of nosocomial infection. We report a structural and functional analysis of the catalytic activity of CD27L against C. difficile and other bacterial strains. We show that truncation of the endolysin to the N-terminal domain, CD27L1-179, gave an increased lytic activity against cells of C. difficile, while the C-terminal region, CD27L180-270, failed to produce lysis. CD27L1-179 also has increased activity against other bacterial species that are targeted by the full-length protein and in addition was able to lyse some CD27L-insensitive strains. However, CD27L1-179 retained a measure of specificity, failing to lyse a wide range of bacteria. The use of green fluorescent protein (GFP)-labeled proteins demonstrated that both CD27L and CD27L1-179 bound to C. difficile cell walls. The crystal structure of CD27L1-179 confirms that the enzyme is a zinc-dependent N-acetylmuramoyl-l-alanine amidase. A structure-based sequence analysis allowed us to identify four catalytic residues, a proton relay cascade, and a substrate binding pocket. A BLAST search shows that the closest-related amidases almost exclusively target Clostridia. This implied that the catalytic domain alone contained features that target a specific bacterial species. To test this hypothesis, we modified Leu 98 to a Trp residue which is found in an endolysin from a bacteriophage of Listeria monocytogenes (PlyPSA). This mutation in CD27L resulted in an increased activity against selected serotypes of L. monocytogenes, demonstrating the potential to tune the species specificity of the catalytic domain of an endolysin.

• Galburt EA, Parrondo JM, Grill SW
• RNA polymerase pushing.
• Biophys Chem. 2011; 157: 43-7
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• Molecular motors can exhibit Brownian ratchet or power stroke mechanisms. These mechanistic categories are related to transition state position: An early transition state suggests that chemical energy is stored and then released during the step (stroke) while a late transition state suggests that the release of chemical energy rectifies thermally activated motion that has already occurred (ratchet). Cellular RNA polymerases are thought to be ratchets that can push each other forward to reduce pausing during elongation. Here, by constructing a two-dimensional energy landscape from the individual landscapes of active and backtracked enzymes, we identify a new pushing mechanism which is the result of a saddle trajectory that arises in the two-dimensional energy landscape of interacting enzymes. We show that this mechanism is more effective with an early transition state suggesting that interacting RNAPs might translocate via a power stroke.

• Bray JK, Weiss DR, Levitt M
• Optimized torsion-angle normal modes reproduce conformational changes more accurately than cartesian modes.
• Biophys J. 2011; 101: 2966-9
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• We present what to our knowledge is a new method of optimized torsion-angle normal-mode analysis, in which the normal modes move along curved paths in Cartesian space. We show that optimized torsion-angle normal modes reproduce protein conformational changes more accurately than Cartesian normal modes. We also show that orthogonalizing the displacement vectors from torsion-angle normal-mode analysis and projecting them as straight lines in Cartesian space does not lead to better performance than Cartesian normal modes. Clearly, protein motion is more naturally described by curved paths in Cartesian space.

• Gleghorn ML, Davydova EK, Basu R, Rothman-Denes LB, Murakami KS
• X-ray crystal structures elucidate the nucleotidyl transfer reaction of transcript initiation using two nucleotides.
• Proc Natl Acad Sci U S A. 2011; 108: 3566-71
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• We have determined the X-ray crystal structures of the pre- and postcatalytic forms of the initiation complex of bacteriophage N4 RNA polymerase that provide the complete set of atomic images depicting the process of transcript initiation by a single-subunit RNA polymerase. As observed during T7 RNA polymerase transcript elongation, substrate loading for the initiation process also drives a conformational change of the O-helix, but only the correct base pairing between the +2 substrate and DNA base is able to complete the O-helix conformational transition. Substrate binding also facilitates catalytic metal binding that leads to alignment of the reactive groups of substrates for the nucleotidyl transfer reaction. Although all nucleic acid polymerases use two divalent metals for catalysis, they differ in the requirements and the timing of binding of each metal. In the case of bacteriophage RNA polymerase, we propose that catalytic metal binding is the last step before the nucleotidyl transfer reaction.

• Martinez-Rucobo FW, Sainsbury S, Cheung AC, Cramer P
• Architecture of the RNA polymerase-Spt4/5 complex and basis of universal transcription processivity.
• EMBO J. 2011; 30: 1302-10
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• Related RNA polymerases (RNAPs) carry out cellular gene transcription in all three kingdoms of life. The universal conservation of the transcription machinery extends to a single RNAP-associated factor, Spt5 (or NusG in bacteria), which renders RNAP processive and may have arisen early to permit evolution of long genes. Spt5 associates with Spt4 to form the Spt4/5 heterodimer. Here, we present the crystal structure of archaeal Spt4/5 bound to the RNAP clamp domain, which forms one side of the RNAP active centre cleft. The structure revealed a conserved Spt5-RNAP interface and enabled modelling of complexes of Spt4/5 counterparts with RNAPs from all kingdoms of life, and of the complete yeast RNAP II elongation complex with bound Spt4/5. The N-terminal NGN domain of Spt5/NusG closes the RNAP active centre cleft to lock nucleic acids and render the elongation complex stable and processive. The C-terminal KOW1 domain is mobile, but its location is restricted to a region between the RNAP clamp and wall above the RNA exit tunnel, where it may interact with RNA and/or other factors.

• Grohmann D et al.
• The initiation factor TFE and the elongation factor Spt4/5 compete for the RNAP clamp during transcription initiation and elongation.
• Mol Cell. 2011; 43: 263-74
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• TFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP "clamp" region as a regulatory hot spot for both transcription initiation and transcription elongation.

• Ulrich S, Kool ET
• Importance of steric effects on the efficiency and fidelity of transcription by T7 RNA polymerase.
• Biochemistry. 2011; 50: 10343-9
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• DNA-dependent RNA polymerases such as T7 RNA polymerase (T7 RNAP) perform the transcription of DNA into mRNA with high efficiency and high fidelity. Although structural studies have provided a detailed account of the molecular basis of transcription, the relative importance of factors like hydrogen bonds and steric effects remains poorly understood. We report herein the first study aimed at systematically probing the importance of steric and electrostatic effects on the efficiency and fidelity of DNA transcription by T7 RNAP. We used synthetic nonpolar analogues of thymine with sizes varying in subangstrom increments to probe the steric requirements of T7 RNAP during the elongation mode of transcription. Enzymatic assays with internal radiolabeling were performed to compare the efficiency of transcription of modified DNA templates with a natural template containing thymine as a reference. Furthermore, we analyzed effects on the fidelity by measuring the composition of RNA transcripts by enzymatic digestion followed by two-dimensional thin layer chromatography separation. Our results demonstrate that hydrogen bonds play an important role in the efficiency of transcription but, interestingly, do not appear to be required for faithful transcription. Steric effects (size and shape variations) are found to be significant both in insertion of a new RNA base and in extension beyond it.

• Surovtseva YV et al.
• Mitochondrial ribosomal protein L12 selectively associates with human mitochondrial RNA polymerase to activate transcription.
• Proc Natl Acad Sci U S A. 2011; 108: 17921-6
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• Basal transcription of human mitochondrial DNA (mtDNA) in vitro requires the single-subunit, bacteriophage-related RNA polymerase, POLRMT, and transcription factor h-mtTFB2. This two-component system is activated differentially at mtDNA promoters by human mitochondrial transcription factor A (h-mtTFA). Mitochondrial ribosomal protein L7/L12 (MRPL12) binds directly to POLRMT, but whether it does so in the context of the ribosome or as a "free" protein in the matrix is unknown. Furthermore, existing evidence that MRPL12 activates mitochondrial transcription derives from overexpression studies in cultured cells and transcription experiments using crude mitochondrial lysates, precluding direct effects of MRPL12 on transcription to be assigned. Here, we report that depletion of MRPL12 from HeLa cells by shRNA results in decreased steady-state levels of mitochondrial transcripts, which are not accounted for by changes in RNA stability. We also show that a significant "free" pool of MRPL12 exists in human mitochondria not associated with ribosomes. "Free" MRPL12 binds selectively to POLRMT in vivo in a complex distinct from those containing h-mtTFB2. Finally, using a fully recombinant mitochondrial transcription system, we demonstrate that MRPL12 stimulates promoter-dependent and promoter-independent transcription directly in vitro. Based on these results, we propose that, when not associated with ribosomes, MRPL12 has a second function in transcription, perhaps acting to facilitate the transition from initiation to elongation. We speculate that this is one mechanism to coordinate mitochondrial ribosome biogenesis and transcription in human mitochondria, where transcription of rRNAs from the mtDNA presumably needs to be adjusted in accordance with the rate of import and assembly of the nucleus-encoded MRPs into ribosomes.

• Hirtreiter A et al.
• Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif.
• Nucleic Acids Res. 2010; 38: 4040-51
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• Spt5 is the only known RNA polymerase-associated factor that is conserved in all three domains of life. We have solved the structure of the Methanococcus jannaschii Spt4/5 complex by X-ray crystallography, and characterized its function and interaction with the archaeal RNAP in a wholly recombinant in vitro transcription system. Archaeal Spt4 and Spt5 form a stable complex that associates with RNAP independently of the DNA-RNA scaffold of the elongation complex. The association of Spt4/5 with RNAP results in a stimulation of transcription processivity, both in the absence and the presence of the non-template strand. A domain deletion analysis reveals the molecular anatomy of Spt4/5--the Spt5 Nus-G N-terminal (NGN) domain is the effector domain of the complex that both mediates the interaction with RNAP and is essential for its elongation activity. Using a mutagenesis approach, we have identified a hydrophobic pocket on the Spt5 NGN domain as binding site for RNAP, and reciprocally the RNAP clamp coiled-coil motif as binding site for Spt4/5.

• Grohmann D, Klose D, Klare JP, Kay CW, Steinhoff HJ, Werner F
• RNA-binding to archaeal RNA polymerase subunits F/E: a DEER and FRET study.
• J Am Chem Soc. 2010; 132: 5954-5
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• RNA polymerases (RNAP) carry out transcription, the first step in the highly regulated process of gene expression. RNAPs are complex multisubunit enzymes, which undergo extensive structural rearrangements during the transcription cycle (initiation-elongation-termination). They accommodate interactions with the nucleic acid scaffold of transcription complexes (template DNA, DNA/RNA hybrid, and nascent RNA) and interact with a plethora of transcription factors. Here we focused on the RNAP-F/E subcomplex, which forms a stable heterodimer that binds the nascent RNA and thereby stimulates the processivity of elongation complexes. We used the pulsed-EPR method DEER and fluorescence spectroscopy to probe for conformational changes within the F/E dimer. Our results demonstrate that, upon binding of RNA, F/E remains in a stable conformation, which suggests that it serves as a structurally rigid guiding rail for the growing RNA chain during transcription.

• Mie M, Sugita R, Endoh T, Kobatake E
• Evaluation of small ligand-protein interactions by using T7 RNA polymerase with DNA-modified ligand.
• Anal Biochem. 2010; 405: 109-13
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• The interaction between proteins and ligands was evaluated by T7 RNA polymerase transcription with a DNA-modified ligand. The principle of this method is suppression of T7 RNA polymerase transcription by binding of a protein to small ligand modified by conjugation with a T7 RNA polymerase promoter. To demonstrate proof of principle, biotin or antifolate methotrexate was modified by covalent attachment of a T7 RNA promoter. Using these T7 RNA promoter-modified ligands, T7 RNA polymerase transcriptions were performed in the presence or absence of an anti-biotin antibody or recombinant human dihydrofolate reductase, respectively. Transcription was suppressed in the presence of each binding protein plus its modified ligand, but not in the absence of the binding protein.

• DeHaseth PL, Gott JM
• Conformational flexibility of sigma(70) in anti-terminator loading.
• Mol Microbiol. 2010; 75: 543-6
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• In promoter DNA, the preferred distance of the -10 and -35 elements for interacting with RNA polymerase-bound sigma(70) is 17 bp. However, the Devi et al. paper in this issue of Molecular Microbiology demonstrates that when the C-terminal domain of sigma(70), including the 3.2 linker, is not attached to the core enzyme, distances between 0 and 3 bp can be accommodated. This attests to the great flexibility of the 3.2 linker. The particularly stable complex with the 2 bp separation may lend itself to structural studies of an early elongation complex containing sigma(70).

• Varadarajan PA, Del Vecchio D
• Design and characterization of a three-terminal transcriptional device through polymerase per second.
• IEEE Trans Nanobioscience. 2009; 8: 281-9
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• In this paper, we provide an in silico input-output characterization of a three-terminal transcriptional device employing polymerase per second (PoPS) as input and output. The device is assembled from well-characterized parts of the bacteriophage lambda switch transcriptional circuit. We draw the analogy between voltage and protein concentration and between current and PoPS to demonstrate that the characteristics of the three-terminal transcriptional device are qualitatively similar to those of a bipolar junction transistor (BJT). In particular, as it occurs in a BJT, the device can be tuned to operate either as a linear amplifier or as a switch. When the device operates as a linear amplifier, gains of twofolds can be obtained, which are considerably smaller than those obtained in a BJT (in which 100-fold amplification gains can be reached). This fact suggests that the parts extracted from natural transcriptional systems may be naturally designed mostly to process and store information as opposed to amplify signals.

• Zaher HS, Green R
• Fidelity at the molecular level: lessons from protein synthesis.
• Cell. 2009; 136: 746-62
• Display abstract

• The faithful and rapid translation of genetic information into peptide sequences is an indispensable property of the ribosome. The mechanistic understanding of strategies used by the ribosome to achieve both speed and fidelity during translation results from nearly a half century of biochemical and structural studies. Emerging from these studies is the common theme that the ribosome uses local as well as remote conformational switches to govern induced-fit mechanisms that ensure accuracy in codon recognition during both tRNA selection and translation termination.

• Rhee AC, Somerlot BH, Parimi N, Gott JM
• Distinct roles for sequences upstream of and downstream from Physarum editing sites.
• RNA. 2009; 15: 1753-65
• Display abstract

• RNAs in the mitochondria of Physarum polycephalum contain nonencoded nucleotides that are added during RNA synthesis. Essentially all steady-state RNAs are accurately and fully edited, yet the signals guiding these precise nucleotide insertions are presently unknown. To localize the regions of the template that are required for editing, we constructed a series of chimeric templates that substitute varying amounts of DNA either upstream of or downstream from C insertion sites. Remarkably, all sequences necessary for C addition are contained within approximately 9 base pairs on either side of the insertion site. In addition, our data strongly suggest that sequences within this critical region affect different steps in the editing reaction. Template alterations upstream of an editing site influence nucleotide selection and/or insertion, while downstream changes affect editing site recognition and templated extension from the added, unpaired nucleotide. The data presented here provide the first evidence that individual regions of the DNA template play discrete mechanistic roles and represent a crucial initial step toward defining the source of the editing specificity in Physarum mitochondria. In addition, these findings have mechanistic implications regarding the potential involvement of the mitochondrial RNA polymerase in the editing reaction.

• Symmons MF, Luisi BF
• Through ancient rings thread programming strings.
• Structure. 2009; 17: 1429-31

• Erie DA, Kennedy SR
• Forks, pincers, and triggers: the tools for nucleotide incorporation and translocation in multi-subunit RNA polymerases.
• Curr Opin Struct Biol. 2009; 19: 708-14
• Display abstract

• The central role of RNA polymerase (RNAP) is to catalyze the processive synthesis of a growing RNA transcript. Recent structural and biophysical data have led to a deeper understanding of the nucleotide addition cycle and insight into the structure-function relationships that govern transcription elongation. In this review, we discuss kinetic data on nucleotide incorporation in the context of crystal structures, which show RNAP in multiple conformations. We present a facilitated Brownian ratchet model of nucleotide incorporation, in which templated NTP binding to a non-catalytic site in the main channel promotes the conformational changes that lead to opening of the catalytic site and translocation.

• Demidenko AA, Nibert ML
• Probing the transcription mechanisms of reovirus cores with molecules that alter RNA duplex stability.
• J Virol. 2009; 83: 5659-70
• Display abstract

• The mammalian reovirus (MRV) genome comprises 10 double-stranded RNA (dsRNA) segments, packaged along with transcriptase complexes inside each core particle. Effects of four small molecules on transcription by MRV cores were studied for this report, chosen for their known capacities to alter RNA duplex stability. Spermidine and spermine, which enhance duplex stability, inhibited transcription, whereas dimethyl sulfoxide and trimethylglycine, which attenuate duplex stability, stimulated transcription. Different mechanisms were identified for inhibition or activation by these molecules. With spermidine, one round of transcription occurred normally, but subsequent rounds were inhibited. Thus, inhibition occurred at the transition between the end of elongation in one round and initiation in the next round of transcription. Dimethyl sulfoxide or trimethylglycine, on the other hand, had no effect on transcription by a constitutively active fraction of cores in each preparation but activated transcription in another fraction that was otherwise silent for the production of elongated transcripts. Activation of this other fraction occurred at the transition between transcript initiation and elongation, i.e., at promoter escape. These results suggest that the relative stability of RNA duplexes is most important for certain steps in the particle-associated transcription cycles of dsRNA viruses and that small molecules are useful tools for probing these and probably other steps.

• Liu X, Martin CT
• Transcription elongation complex stability: the topological lock.
• J Biol Chem. 2009; 284: 36262-70
• Display abstract

• Transcription machinery from a variety of organisms shows striking mechanistic similarity. Both multi- and single subunit RNA polymerases have evolved an 8-10-base pair RNA-DNA hybrid as a part of a stably transcribing elongation complex. Through characterization of halted complexes that can readily carry out homopolymeric slippage synthesis, this study reveals that T7 RNA polymerase elongation complexes containing only a 4-base pair hybrid can nevertheless be more stable than those with the normal 8-base pair hybrid. We propose that a key feature of this stability is the topological threading of RNA through the complex and/or around the DNA template strand. The data are consistent with forward translocation as a mechanism to allow unthreading of the topological lock, as can occur during programmed termination of transcription.

• Gleghorn ML, Davydova EK, Rothman-Denes LB, Murakami KS
• Structural basis for DNA-hairpin promoter recognition by the bacteriophage N4 virion RNA polymerase.
• Mol Cell. 2008; 32: 707-17
• Display abstract

• Coliphage N4 virion-encapsidated RNA polymerase (vRNAP) is a member of the phage T7-like single-subunit RNA polymerase (RNAP) family. Its central domain (mini-vRNAP) contains all RNAP functions of the full-length vRNAP, which recognizes a 5 to 7 base pair stem and 3 nucleotide loop hairpin DNA promoter. Here, we report the X-ray crystal structures of mini-vRNAP bound to promoters. Mini-vRNAP uses four structural motifs to recognize DNA sequences at the hairpin loop and stem and to unwind DNA. Despite their low sequence similarity, three out of four motifs are shared with T7 RNAP that recognizes a double-stranded DNA promoter. The binary complex structure and results of engineered disulfide linkage experiments reveal that the plug and motif B loop, which block the access of template DNA to the active site in the apo-form mini-vRNAP, undergo a large-scale conformational change upon promoter binding, explaining the restricted promoter specificity that is critical for N4 phage early transcription.

• Campagnola G, Weygandt M, Scoggin K, Peersen O
• Crystal structure of coxsackievirus B3 3Dpol highlights the functional importance of residue 5 in picornavirus polymerases.
• J Virol. 2008; 82: 9458-64
• Display abstract

• The crystal structure of the coxsackievirus B3 polymerase has been solved at 2.25-A resolution and is shown to be highly homologous to polymerases from poliovirus, rhinovirus, and foot-and-mouth disease viruses. Together, these structures highlight several conserved structural elements in picornaviral polymerases, including a proteolytic activation-dependent N-terminal structure that is essential for full activity. Interestingly, a comparison of all of the picornaviral polymerase structures shows an unusual conformation for residue 5, which is always located at a distortion in the beta-strand composed of residues 1 to 8. In our earlier structure of the poliovirus polymerase, we attributed this conformation to a crystal packing artifact, but the observation that this conformation is conserved among picornaviruses led us to examine the role of this residue in further detail. Here we use coxsackievirus polymerase to show that elongation activity correlates with the hydrophobicity of residue 5 and, surprisingly, more hydrophobic residues result in higher activity. Based on structural analysis, we propose that this residue becomes buried during the nucleotide repositioning step that occurs prior to phosphoryl transfer. We present a model in which the buried N terminus observed in all picornaviral polymerases is essential for stabilizing the structure during this conformational change.

• Limanskaia OIu, Limanskii AP
• [Visualization of elongation complexes for t7 Rna polymerase by atomic force microscopy].
• Mol Biol (Mosk). 2008; 42: 533-42
• Display abstract

• Complexes of bacteriophage T7 RNA polymerase (RNAP) with a DNA template for transcription elongation were visualized by atomic force microscopy (AFM). Fragment of pGEMEX linear DNA with length of 1414 bp carrying promot- er and terminator of bacteriophage T7 was DNA template for transcription. Promoter and terminator are located unsymmetrically on the ends of DNA template. Images of stable complexes of T7 RNAP with terminal fragments of DNA template were obtained for single molecules. Complexes of a single DNA template molecule with 2-3 T7 RNAP molecules corresponding to stages of initiation, elongation and termination of transcription were visualized under the elimination of non-specific DNA-protein binding. Also complexes of DNA, RNAP and RNA transcripts were imaged. Our results suggest that the initial stage is the formation of complex between T7 RNAP and terminal fragment of DNA template. Because promoter is localized near DNA terminus, it makes impossible an ommision of the promoter site.

• Hirata A, Klein BJ, Murakami KS
• The X-ray crystal structure of RNA polymerase from Archaea.
• Nature. 2008; 451: 851-4
• Display abstract

• The transcription apparatus in Archaea can be described as a simplified version of its eukaryotic RNA polymerase (RNAP) II counterpart, comprising an RNAPII-like enzyme as well as two general transcription factors, the TATA-binding protein (TBP) and the eukaryotic TFIIB orthologue TFB. It has been widely understood that precise comparisons of cellular RNAP crystal structures could reveal structural elements common to all enzymes and that these insights would be useful in analysing components of each enzyme that enable it to perform domain-specific gene expression. However, the structure of archaeal RNAP has been limited to individual subunits. Here we report the first crystal structure of the archaeal RNAP from Sulfolobus solfataricus at 3.4 A resolution, completing the suite of multi-subunit RNAP structures from all three domains of life. We also report the high-resolution (at 1.76 A) crystal structure of the D/L subcomplex of archaeal RNAP and provide the first experimental evidence of any RNAP possessing an iron-sulphur (Fe-S) cluster, which may play a structural role in a key subunit of RNAP assembly. The striking structural similarity between archaeal RNAP and eukaryotic RNAPII highlights the simpler archaeal RNAP as an ideal model system for dissecting the molecular basis of eukaryotic transcription.

• Meyer PR, Rutvisuttinunt W, Matsuura SE, So AG, Scott WA
• Stable complexes formed by HIV-1 reverse transcriptase at distinct positions on the primer-template controlled by binding deoxynucleoside triphosphates or foscarnet.
• J Mol Biol. 2007; 369: 41-54
• Display abstract

• Binding of the next complementary dNTP by the binary complex containing HIV-1 reverse transcriptase (RT) and primer-template induces conformational changes that have been implicated in catalytic function of RT. We have used DNase I footprinting, gel electrophoretic mobility shift, and exonuclease protection assays to characterize the interactions between HIV-1 RT and chain-terminated primer-template in the absence and presence of various ligands. Distinguishable stable complexes were formed in the presence of foscarnet (an analog of pyrophosphate), the dNTP complementary to the first (+1) templating nucleotide or the dNTP complementary to the second (+2) templating nucleotide. The position of HIV-1 RT on the primer-template in each of these complexes is different. RT is located upstream in the foscarnet complex, relative to the +1 complex, and downstream in the +2 complex. These results suggest that HIV-1 RT can translocate along the primer-template in the absence of phosphodiester bond formation. The ability to form a specific foscarnet complex might explain the inhibitory properties of this compound. The ability to recognize the second templating nucleotide has implications for nucleotide misincorporation.

• Kennedy WP, Momand JR, Yin YW
• Mechanism for de novo RNA synthesis and initiating nucleotide specificity by t7 RNA polymerase.
• J Mol Biol. 2007; 370: 256-68
• Display abstract

• DNA-directed RNA polymerases are capable of initiating synthesis of RNA without primers, the first catalytic stage of initiation is referred to as de novo RNA synthesis. De novo synthesis is a unique phase in the transcription cycle where the RNA polymerase binds two nucleotides rather than a nascent RNA polymer and a single nucleotide. For bacteriophage T7 RNA polymerase, transcription begins with a marked preference for GTP at the +1 and +2 positions. We determined the crystal structures of T7 RNA polymerase complexes captured during the de novo RNA synthesis. The DNA substrates in the structures in the complexes contain a common Phi 10 duplex promoter followed by a unique five base single-stranded extension of template DNA whose sequences varied at positions +1 and +2, thereby allowing for different pairs of initiating nucleotides GTP, ATP, CTP or UTP to bind. The structures show that the initiating nucleotides bind RNA polymerase in locations distinct from those described previously for elongation complexes. Selection bias in favor of GTP as an initiating nucleotide is accomplished by shape complementarity, extensive protein side-chain and strong base-stacking interactions for the guanine moiety in the enzyme active site. Consequently, an initiating GTP provides the largest stabilization force for the open promoter conformation.

• Kashkina E et al.
• Multisubunit RNA polymerases melt only a single DNA base pair downstream of the active site.
• J Biol Chem. 2007; 282: 21578-82
• Display abstract

• To extend the nascent transcript, RNA polymerases must melt the DNA duplex downstream from the active site to expose the next acceptor base for substrate binding and incorporation. A number of mechanisms have been proposed to account for the manner in which the correct substrate is selected, and these differ in their predictions as to how far the downstream DNA is melted. Using fluorescence quenching experiments, we provide evidence that cellular RNA polymerases from bacteria and yeast melt only one DNA base pair downstream from the active site. These data argue against a model in which multiple NTPs are lined up downstream of the active site.

• Wu T, Schwartz DC
• Transchip: single-molecule detection of transcriptional elongation complexes.
• Anal Biochem. 2007; 361: 31-46
• Display abstract

• A new single-molecule system, Transchip, was developed for analysis of transcription products at their genomic origins. The bacteriophage T7 RNA polymerase and its promoters were used in a model system, and resultant RNAs were imaged and detected at their positions along single template DNA molecules. The Transchip system has drawn from critical aspects of Optical Mapping, a single-molecule system that enables the construction of high-resolution ordered restriction maps of whole genomes from single DNA molecules. Through statistical analysis of hundreds of single-molecule template/transcript complexes, Transchip enables analysis of the locations and strength of promoters, the direction and processivity of transcription reactions, and the termination of transcription. These novel results suggest that the new system may serve as a high-throughput platform to investigate transcriptional events on a large genome-wide scale.

• Altafaj X, Joux N, Ronjat M, De Waard M
• Oocyte expression with injection of purified T7 RNA polymerase.
• Methods Mol Biol. 2006; 322: 55-67
• Display abstract

• The Xenopus oocyte is a widely used system for protein expression. Investigators have had the choice between two different techniques: injection into the cytoplasm of in vitro transcribed complementary RNA (cRNA) or injection into the nucleus of complementary DNA (cDNA). We report on a third expression technique that is based on the combined injection of cDNA and purified T7 RNA polymerase directly into the cytoplasm of oocytes.

• Zhou Y, Martin CT
• Observed instability of T7 RNA polymerase elongation complexes can be dominated by collision-induced "bumping".
• J Biol Chem. 2006; 281: 24441-8
• Display abstract

• T7 RNA polymerase elongates RNA at a relatively high rate and can displace many tightly bound protein-DNA complexes. Despite these properties, measurements of the stability of stalled elongation complexes have shown lifetimes that are much shorter than those of the multisubunit RNA polymerases. In this work, we demonstrate that the apparent instability of stalled complexes actually arises from the action of trailing RNA polymerases (traveling in the same direction) displacing the stalled complex. Moreover, the instability caused by collision between two polymerases is position dependent. A second polymerase is blocked from promoter binding when a leading complex is stalled 12 bp or less from the promoter. The trailing complex can bind and make abortive transcripts when the leading complex is between 12 and 20 bp from the promoter, but it cannot displace the first complex since it is in a unstable initiation conformation. Only when the leading complex is stalled more than 20 bp away from the promoter can a second polymerase bind, initiate, and displace the leading complex.

• Gong P, Martin CT
• Mechanism of instability in abortive cycling by T7 RNA polymerase.
• J Biol Chem. 2006; 281: 23533-44
• Display abstract

• Abortive transcription, the premature release of short transcripts 2-8 bases in length, is a unique feature of transcription, accompanying the transition from initiation to elongation in all RNA polymerases. The current study focuses on major factors that relate to the stability of initially transcribing abortive complexes in T7 RNA polymerase. Building on previous studies, results reveal that collapse of the DNA from the downstream end of the bubble is a major contributor to the characteristic instability of abortive complexes. Furthermore, transcription from a novel DNA construct containing a nick between positions -14 and -13 of the nontemplate strand suggests that the more flexible promoter reduces somewhat the strain inherent in initially transcribing complexes, with a resulting decrease in abortive product release. Finally, as assessed by exonuclease III footprinting and transcription profiles, a DNA construct defective in bubble collapse specifically from the downstream end exhibits less abortive cycling and little perturbation of the final transition to elongation, including the process of promoter release.

• Zhang H, Rhee C, Bebenek A, Drake JW, Wang J, Konigsberg W
• The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination.
• Biochemistry. 2006; 45: 2211-20
• Display abstract

• Several variants of RB69 DNA polymerase (RB69 pol) with single-site replacements in the nascent base-pair binding pocket are less discriminating with respect to noncomplementary dNMP incorporation than the wild-type enzyme. To quantify the loss in base selectivity, we determined the transient-state kinetic parameters for incorporation of correct and all combinations of incorrect dNMPs by the exonuclease-deficient form of one of these RB69 pol variants, L561A, using rapid chemical quench assays. The L561A variant did not significantly alter the k(pol) and K(D) values for incorporation of correct dNMPs, but it showed increased incorporation efficiency (k(pol)/K(D)) for mispaired bases relative to the wild-type enzyme. The incorporation efficiency for mispaired bases by the L561A variant ranged from 1.5 x 10(-)(5) microM(-)(1) s(-)(1) for dCMP opposite templating C to 2 x 10(-)(3) microM(-)(1) s(-)(1) for dAMP opposite templating C. These k(pol)/K(D) values are 3-60-fold greater than those observed with the wild-type enzyme. The effect of the L561A replacement on the mutation frequency in vivo was determined by infecting Escherichia coli harboring a plasmid encoding the L561A variant of RB69 pol with T4 phage bearing a mutant rII locus, and the rates of reversions to rII(+) were scored. The exonuclease-proficient RB69 pol L561A displayed a weak mutator phenotype. In contrast, no progeny phage were produced after infection of E. coli, expressing an exonuclease-deficient RB69 pol L561A, with either mutant or wild-type T4 phage. This dominant-lethal phenotype was attributed to error catastrophe caused by the high rate of mutation expected from combining the pol L561A and exo(-) mutator activities.

• Zlatanova J, McAllister WT, Borukhov S, Leuba SH
• Single-molecule approaches reveal the idiosyncrasies of RNA polymerases.
• Structure. 2006; 14: 953-66
• Display abstract

• Recently developed single-molecule techniques have provided new insights into the function of one of the most complex and highly regulated processes in the cell--the transcription of the DNA template into RNA. This review discusses methods and results from this emerging field, and it puts them in perspective of existing biochemical and structural data.

• Kapanidis AN, Margeat E, Ho SO, Kortkhonjia E, Weiss S, Ebright RH
• Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism.
• Science. 2006; 314: 1144-7
• Display abstract

• Using fluorescence resonance energy transfer to monitor distances within single molecules of abortively initiating transcription initiation complexes, we show that initial transcription proceeds through a "scrunching" mechanism, in which RNA polymerase (RNAP) remains fixed on promoter DNA and pulls downstream DNA into itself and past its active center. We show further that putative alternative mechanisms for RNAP active-center translocation in initial transcription, involving "transient excursions" of RNAP relative to DNA or "inchworming" of RNAP relative to DNA, do not occur. The results support a model in which a stressed intermediate, with DNA-unwinding stress and DNA-compaction stress, is formed during initial transcription, and in which accumulated stress is used to drive breakage of interactions between RNAP and promoter DNA and between RNAP and initiation factors during promoter escape.

• Tuske S et al.
• Inhibition of bacterial RNA polymerase by streptolydigin: stabilization of a straight-bridge-helix active-center conformation.
• Cell. 2005; 122: 541-52
• Display abstract

• We define the target, mechanism, and structural basis of inhibition of bacterial RNA polymerase (RNAP) by the tetramic acid antibiotic streptolydigin (Stl). Stl binds to a site adjacent to but not overlapping the RNAP active center and stabilizes an RNAP-active-center conformational state with a straight-bridge helix. The results provide direct support for the proposals that alternative straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations exist and that cycling between straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations is required for RNAP function. The results set bounds on models for RNAP function and suggest strategies for design of novel antibacterial agents.

• Hansen AM et al.
• Structural basis for the function of stringent starvation protein a as a transcription factor.
• J Biol Chem. 2005; 280: 17380-91
• Display abstract

• Stringent starvation protein A (SspA) of Escherichia coli is an RNA polymerase-associated transcriptional activator for the lytic development of phage P1 and is essential for stationary phase-induced acid tolerance of E. coli. We report the crystal structure of Yersinia pestis SspA, which is 83% identical to E. coli SspA in amino acid sequence and is functionally complementary in supporting the lytic growth of phage P1 and acid resistance of an E. coli sspA mutant. The structure reveals that SspA assumes the characteristic fold of glutathione S-transferase (GST). However, SspA lacks GST activity and does not bind glutathione. Three regions of SspA are flexible, the N and C termini and the alpha2-helix. The structure also reveals a conserved surface-exposed pocket composed of residues from a loop between helices alpha3 and alpha4. The functional roles of these structural features were investigated by assessing the ability of deletion and site-directed mutants to confer acid resistance of E. coli and to activate transcription from a phage P1 late promoter, thereby supporting the lytic growth of phage P1. The results indicate that the flexible regions are not critical for SspA function, whereas the surface pocket is important for both transcriptional activation of the phage P1 late promoter and acid resistance of E. coli. The size, shape, and property of the pocket suggest that it mediates protein-protein interactions. SspA orthologs from Y. pestis, Vibrio cholerae, and Pseudomonas aeruginosa are all functional in acid resistance of E. coli, whereas only Y. pestis SspA supports phage P1 growth.

• Ma K, Temiakov D, Anikin M, McAllister WT
• Probing conformational changes in T7 RNA polymerase during initiation and termination by using engineered disulfide linkages.
• Proc Natl Acad Sci U S A. 2005; 102: 17612-7
• Display abstract

• During the transition from an initiation complex to an elongation complex (EC), the single-subunit bacteriophage T7 RNA polymerase (RNAP) undergoes dramatic conformational changes. To explore the significance of these changes, we constructed mutant RNAPs that are able to form disulfide bonds that limit the mobility of elements that are involved in the transition (or its reversal) and examined the effects of the crosslinks on initiation and termination. A crosslink that is specific to the initiation complex conformation blocks transcription at 5-6 nt, presumably by preventing isomerization to an EC. A crosslink that is specific to the EC conformation has relatively little effect on elongation or on termination at a class I terminator (T), which involves the formation of a stable stem-loop structure in the RNA. Crosslinked ECs also pause and resume transcription normally at a class II pause site (concatamer junction) but are deficient in termination at a class II terminator (PTH, which is found in human preparathyroid hormone gene), both of which involve a specific recognition sequence. The crosslinked amino acids in the EC lie close to the upstream end of the RNA-DNA hybrid and may prevent a movement of the polymerase that would assist in displacing or releasing RNA from a relatively unstable DNA-RNA hybrid in the paused PTH complex.

• Appleby TC et al.
• Crystal structure of complete rhinovirus RNA polymerase suggests front loading of protein primer.
• J Virol. 2005; 79: 277-88
• Display abstract

• Picornaviruses utilize virally encoded RNA polymerase and a uridylylated protein primer to ensure replication of the entire viral genome. The molecular details of this mechanism are not well understood due to the lack of structural information. We report the crystal structure of human rhinovirus 16 3D RNA-dependent RNA polymerase (HRV16 3Dpol) at a 2.4-A resolution, representing the first complete polymerase structure from the Picornaviridae family. HRV16 3Dpol shares the canonical features of other known polymerase structures and contains an N-terminal region that tethers the fingers and thumb subdomains, forming a completely encircled active site cavity which is accessible through a small tunnel on the backside of the molecule. The small thumb subdomain contributes to the formation of a large cleft on the front face of the polymerase which also leads to the active site. The cleft appears large enough to accommodate a template:primer duplex during RNA elongation or a protein primer during the uridylylation stage of replication initiation. Based on the structural features of HRV16 3Dpo1 and the catalytic mechanism known for all polymerases, a front-loading model for uridylylation is proposed.

• Kato M, Ito T, Wagner G, Ellenberger T
• A molecular handoff between bacteriophage T7 DNA primase and T7 DNA polymerase initiates DNA synthesis.
• J Biol Chem. 2004; 279: 30554-62
• Display abstract

• The T7 DNA primase synthesizes tetraribonucleotides that prime DNA synthesis by T7 DNA polymerase but only on the condition that the primase stabilizes the primed DNA template in the polymerase active site. We used NMR experiments and alanine scanning mutagenesis to identify residues in the zinc binding domain of T7 primase that engage the primed DNA template to initiate DNA synthesis by T7 DNA polymerase. These residues cover one face of the zinc binding domain and include a number of aromatic amino acids that are conserved in bacteriophage primases. The phage T7 single-stranded DNA-binding protein gp2.5 specifically interfered with the utilization of tetraribonucleotide primers by interacting with T7 DNA polymerase and preventing a productive interaction with the primed template. We propose that the opposing effects of gp2.5 and T7 primase on the initiation of DNA synthesis reflect a sequence of mutually exclusive interactions that occur during the recycling of the polymerase on the lagging strand of the replication fork.

• Stano NM, Patel SS
• T7 lysozyme represses T7 RNA polymerase transcription by destabilizing the open complex during initiation.
• J Biol Chem. 2004; 279: 16136-43
• Display abstract

• Bacteriophage T7 lysozyme binds to T7 RNA polymerase and inhibits transcription initiation and the transition from initiation to elongation. We have investigated each step of transcription initiation to determine where T7 lysozyme has the most effect. Stopped flow and equilibrium DNA binding studies indicate that T7 lysozyme does not inhibit the formation of the preinitiation open complex (open complex in the absence of initiating nucleotide). T7 lysozyme, however, does prevent the formation of a fully open initiation complex (open complex in the presence of the initiating nucleotide). This is consistent with the results that in the presence of T7 lysozyme the rate of G ladder RNA synthesis is about 5-fold slower and the GTP Kd is about 2-fold higher, but T7 lysozyme does not inhibit the initial rate of RNA synthesis with a premelted bulge-6 promoter (bubble from -4 to +2). Neither the RNA synthesis rate nor the extent of promoter opening is restored by increasing the initiating nucleotide concentration, indicating that T7 lysozyme represses transcription by interfering with the formation of a stable and a fully open initiation bubble or by altering the structure of the DNA in the initiation complex. As a consequence of the unstable initiation bubble and/or the inhibition of the conformational changes in the N-terminal domain of T7 RNAP, T7 lysozyme causes an increased production of abortive products from 2- to 5-mer that delays the transition from the initiation to the elongation phase.

• Temiakov D, Patlan V, Anikin M, McAllister WT, Yokoyama S, Vassylyev DG
• Structural basis for substrate selection by t7 RNA polymerase.
• Cell. 2004; 116: 381-91
• Display abstract

• The mechanism by which nucleotide polymerases select the correct substrate is of fundamental importance to the fidelity of DNA replication and transcription. During the nucleotide addition cycle, pol I DNA polymerases undergo the transition from a catalytically inactive "open" to an active "closed" conformation. All known determinants of substrate selection are associated with the "closed" state. To elucidate if this mechanism is conserved in homologous single subunit RNA polymerases (RNAPs), we have determined the structure of T7 RNAP elongation complex with the incoming substrate analog. Surprisingly, the substrate specifically binds to RNAP in the "open" conformation, where it is base paired with the acceptor template base, while Tyr639 provides discrimination of ribose versus deoxyribose substrates. The structure therefore suggests a novel mechanism, in which the substrate selection occurs prior to the isomerization to the catalytically active conformation. Modeling of multisubunit RNAPs suggests that this mechanism might be universal for all RNAPs.

• Westover KD, Bushnell DA, Kornberg RD
• Structural basis of transcription: separation of RNA from DNA by RNA polymerase II.
• Science. 2004; 303: 1014-6
• Display abstract

• The structure of an RNA polymerase II-transcribing complex has been determined in the posttranslocation state, with a vacancy at the growing end of the RNA-DNA hybrid helix. At the opposite end of the hybrid helix, the RNA separates from the template DNA. This separation of nucleic acid strands is brought about by interaction with a set of proteins loops in a strand/loop network. Formation of the network must occur in the transition from abortive initiation to promoter escape.

• Kingston RL, Hamel DJ, Gay LS, Dahlquist FW, Matthews BW
• Structural basis for the attachment of a paramyxoviral polymerase to its template.
• Proc Natl Acad Sci U S A. 2004; 101: 8301-6
• Display abstract

• The nucleocapsid of measles virus is the template for viral RNA synthesis and is generated through packaging of the genomic RNA by the nucleocapsid protein (N). The viral polymerase associates with the nucleocapsid through a small, trihelical binding domain at the carboxyl terminus of the phosphoprotein (P). Translocation of the polymerase along the nucleocapsid during RNA synthesis is thought to involve the repeated attachment and release of the binding domain. We have investigated the interaction between the binding domain from measles P (amino acids 457-507) and the sequence it recognizes within measles N (amino acids 477-505). By using both solution NMR spectroscopy and x-ray crystallography, we show that N(487-503) binds as a helix to the surface created by the second (alpha2) and third (alpha3) helices of P(457-507), in an orientation parallel to the helix alpha3, creating a four-helix bundle. The binding interface is tightly packed and dominated by hydrophobic amino acids. Binding and folding of N(487-503) are coupled. However, when not bound to P, N(487-503) does not resemble a statistical random coil but instead exists in a loosely structured state that mimics the bound conformation. We propose that before diffusional encounter, the ensemble of accessible conformations for N(487-503) is biased toward structures capable of binding P, facilitating rapid association of the two proteins. This study provides a structural analysis of polymerase-template interactions in a paramyxovirus and presents an example of a protein-protein interaction that must be only transiently maintained as part of its normal function.

• Kuzmine I, Gottlieb PA, Martin CT
• Binding of the priming nucleotide in the initiation of transcription by T7 RNA polymerase.
• J Biol Chem. 2003; 278: 2819-23
• Display abstract

• Unlike DNA polymerases, an RNA polymerase must initiate transcription de novo, that is binding of the initiating (+1) nucleoside triphosphate must be achieved without benefit of the cooperative binding energetics of an associated primer. Since a single Watson-Crick base pair is not stable in solution, RNA polymerases might be expected to provide additional stabilizing interactions to facilitate binding and positioning of the initiating (priming) nucleoside triphosphate at position +1. Consistent with base-specific stabilizing interactions, of the 17 T7 RNA polymerase promoters in the phage genome, 15 begin with guanine. In this work, we demonstrate that the purine N-7 is important in the utilization of the initial substrate GTP. The fact that on a template encoding AG as the first two bases in the transcript (as in the remaining two of the T7 genome) transcription starts predominantly (but not exclusively) at the G at position +2 additionally implicates the purine O-6 as an important recognition element in the major groove. Finally, results suggest that these interactions serve primarily to position the initiating base in the active site. It is proposed that T7 RNA polymerase interacts directly with the Hoogsteen side of the initial priming GTP (most likely via an interaction with an arginine side chain in the protein) to provide the extra stability required at this unique step in transcription.

• Sousa R, Mukherjee S
• T7 RNA polymerase.
• Prog Nucleic Acid Res Mol Biol. 2003; 73: 1-41

• Kukarin A, Rong M, McAllister WT
• Exposure of T7 RNA polymerase to the isolated binding region of the promoter allows transcription from a single-stranded template.
• J Biol Chem. 2003; 278: 2419-24
• Display abstract

• While the binding region of the T7 promoter must be double-stranded (ds) to function, the non-template strand in the initiation region is dispensable, and a promoter that lacks this element allows efficient initiation. To determine whether the binding region serves merely to recruit the RNA polymerase (RNAP) to the vicinity of a melted initiation region or provides other functions, we utilized a GAL4-T7 RNAP fusion protein to provide an independent binding capacity to the RNAP. When the GAL4-T7 RNAP was recruited to a single-stranded (ss) promoter via a nearby Gal4 recognition sequence, no transcription was observed. However, transcription from the ss promoter could be activated by the addition, in trans, of a ds hairpin loop that contains only the binding region of the promoter. The same results were obtained in the absence of the GAL4 recognition sequence in the template and were also observed with wild type enzyme. Gel-shift experiments indicate that exposure of the RNAP to the isolated binding region facilitates recruitment of the ss template, but that the binding region is displaced from the complex prior to initiation. We conclude that exposure of the RNAP to the isolated binding region reorganizes the enzyme, allowing it to bind to the ss template. These findings have potential implications with regard to mechanisms of promoter binding and melting.

• Liu D, Windsor WT, Wyss DF
• Double-stranded DNA-induced localized unfolding of HCV NS3 helicase subdomain 2.
• Protein Sci. 2003; 12: 2757-67
• Display abstract

• The NS3 helicase of the hepatitis C virus (HCV) unwinds double-stranded (ds) nucleic acid (NA) in an NTP-dependent fashion. Mechanistic details of this process are, however, largely unknown for the HCV helicase. We have studied the binding of dsDNA to an engineered version of subdomain 2 of the HCV helicase (d(2Delta)NS3h) by NMR and circular dichroism. Binding of dsDNA to d(2Delta)NS3h induces a local unfolding of helix (alpha(3)), which includes residues of conserved helicase motif VI (Q(460)RxxRxxR(467)), and strands (beta(1) and beta(8)) from the central beta-sheet. This also occurs upon lowering the pH (4.4) and introducing an R461A point mutation, which disrupt salt bridges with Asp 412 and Asp 427 in the protein structure. NMR studies on d(2Delta)NS3h in the partially unfolded state at low pH map the dsDNA binding site to residues previously shown to be involved in single-stranded DNA binding. Sequence alignment and structural comparison suggest that these Arg-Asp interactions are highly conserved in SF2 DEx(D/H) proteins. Thus, modulation of these interactions by dsNA may allow SF2 helicases to switch between conformations required for helicase function.

• Tunitskaya VL, Kochetkov SN
• Structural-functional analysis of bacteriophage T7 RNA polymerase.
• Biochemistry (Mosc). 2002; 67: 1124-35
• Display abstract

• This review summarizes our results of the structural and functional studies of bacteriophage T7 DNA-dependent RNA polymerase (T7 RNAP). Particular features of this enzyme (the single-subunit composition, relatively low molecular weight) make it the most convenient model for investigating the physicochemical aspects of transcription. The review discusses the main properties of T7 RNAP, interaction between the enzyme and promoter, principle stages of T7-transcription, and also the results of structural and functional studies by affinity modification and both random and site-directed mutagenesis techniques.

• Nickels BE, Roberts CW, Sun H, Roberts JW, Hochschild A
• The sigma(70) subunit of RNA polymerase is contacted by the (lambda)Q antiterminator during early elongation.
• Mol Cell. 2002; 10: 611-22
• Display abstract

• The Q protein of bacteriophage lambda is a transcription antiterminator that modifies the elongation properties of E. coli RNA polymerase (RNAP). To do this, DNA-bound (lambda)Q must first engage a paused elongation complex. Here we show that this engagement of (lambda)Q with RNAP involves an interaction between (lambda)Q and sigma(70), demonstrating that sigma(70) can be a target of regulation during elongation. Furthermore, we provide evidence that this interaction between (lambda)Q and sigma(70) stabilizes a conformation of RNAP that requires the disengagement of a segment of sigma(70) from the core enzyme. Recent structure-based models posit that the transition from the initiation to the elongation phase of transcription involves the staged displacement of sigma(70) from the RNAP core. Our findings provide support for this proposal.

• Johnson RS, Chester RE
• UTP allosterically regulates transcription by Escherichia coli RNA polymerase from the bacteriophage T7 A1 promoter.
• J Mol Biol. 2002; 318: 305-20
• Display abstract

• In the case of Escherichia coli RNA polymerase, UTP at elevated concentrations suppresses terminated transcript accumulation during multiple-round transcription from a DNA construct containing the T7 A1 promoter and T(e) terminator. The step that is affected by UTP at elevated concentrations is promoter clearance. In an attempt to understand better the mechanism by which UTP regulates this step, we analyzed the effect of UTP on the formation of pppApU in the presence of only UTP and ATP. At elevated concentrations, UTP is a non-competitive inhibitor with respect to ATP in the formation of pppApU. This indicates that the effect of UTP on the formation of pppApU is mediated through an allosteric site. Moreover, the magnitude of the inhibition of pppApU formation is sufficient to account for the decrease in terminated transcript accumulation at elevated UTP concentrations. Thus, it appears that UTP modulates terminated transcript accumulation during multiple-round transcription from this DNA construct by allosteric regulation of promoter clearance at the point of transcription initiation.

• Tahirov TH et al.
• Structure of a T7 RNA polymerase elongation complex at 2.9 A resolution.
• Nature. 2002; 420: 43-50
• Display abstract

• The single-subunit bacteriophage T7 RNA polymerase carries out the transcription cycle in an identical manner to that of bacterial and eukaryotic multisubunit enzymes. Here we report the crystal structure of a T7 RNA polymerase elongation complex, which shows that incorporation of an 8-base-pair RNA-DNA hybrid into the active site of the enzyme induces a marked rearrangement of the amino-terminal domain. This rearrangement involves alternative folding of about 130 residues and a marked reorientation (about 130 degrees rotation) of a stable core subdomain, resulting in a structure that provides elements required for stable transcription elongation. A wide opening on the enzyme surface that is probably an RNA exit pathway is formed, and the RNA-DNA hybrid is completely buried in a newly formed, deep protein cavity. Binding of 10 base pairs of downstream DNA is stabilized mostly by long-distance electrostatic interactions. The structure implies plausible mechanisms for the various phases of the transcription cycle, and reveals important structural similarities with the multisubunit RNA polymerases.

• Jayarajah CN, Thompson M
• Signaling of transcriptional chemistry in the on-line detection format.
• Biosens Bioelectron. 2002; 17: 159-71
• Display abstract

• A critical analysis of optical and acoustic wave instrumentation for examining the transcription apparatus and its regulation is given in the present review. The physico-chemical parameters derived from such in vitro experiments are important from a biophysical standpoint. The overall mechanism of transcription is composed of several mechanisms such as DNA-binding and promoter selection, closed and open polymerase complex formation, initiation of RNA synthesis, elongation and termination. Surface plasmon resonance (SPR) and fluorescence spectroscopy are widely employed techniques for investigating these mechanisms in real time. Although the binding of nucleotides, transcription factors (TFs) and inhibitors to RNA polymerase (RNAP) and the DNA template have been studied extensively, the synthesis of mRNA has not been investigated in detail except by methods based on electrophoresis. The use of acoustic wave physics for investigating transcriptional chemistry offers not only a time-course analysis but also the potential to gain insight into structural changes that occur during the process.

• Griko Y, Sreerama N, Osumi-Davis P, Woody RW, Woody AY
• Thermal and urea-induced unfolding in T7 RNA polymerase: calorimetry, circular dichroism and fluorescence study.
• Protein Sci. 2001; 10: 845-53
• Display abstract

• Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme. T7RNAP is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains. Interactions between these structural domains and their stability strongly depend on solvent conditions. The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased beta-sheet and reduced alpha-helix content relative to the native state. Urea-induced unfolding at 25 degrees C reveals a two-step process. The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea. The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows little temperature dependence from 15 degrees to 60 degrees C. The second transition leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation. Near-ultraviolet CD spectra at 25 degrees C at varying concentrations of urea are consistent with this picture. Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding.

• Ohtsuki T, Kimoto M, Ishikawa M, Mitsui T, Hirao I, Yokoyama S
• Unnatural base pairs for specific transcription.
• Proc Natl Acad Sci U S A. 2001; 98: 4922-5
• Display abstract

• An unnatural base pair of 2-amino-6-(N,N-dimethylamino)purine (designated as x) and pyridin-2-one (designated as y) has been developed for specific transcription. The ribonucleoside triphosphates of y and a modified y, 5-methylpyridin-2-one, are selectively incorporated into RNA opposite x in the templates by T7 RNA polymerase. In addition, the sequences of the DNA templates containing x can be confirmed by a dideoxynucleotide chain-terminator method supplemented with the deoxynucleoside triphosphate of y. The bulky dimethylamino group of x in the templates effectively eliminates noncognate pairing with the natural bases. These results enable RNA biosynthesis for the specific incorporation of unnatural nucleotides at the desired positions.

• Best AA, Olsen GJ
• Similar subunit architecture of archaeal and eukaryal RNA polymerases.
• FEMS Microbiol Lett. 2001; 195: 85-90
• Display abstract

• Protein interactions among RNA polymerase small subunits from the archaeon Methanococcus jannaschii were investigated using affinity pulldown assays in pairwise and higher-order combinations. In the most extensive study of archaeal RNA polymerase subunit interactions to date, including 37 pairs of proteins, 10 ternary combinations, and three quaternary combinations, we found evidence for pairwise interactions of subunit D with subunits L and N, and a ternary complex of subunits D, L and N. No other small subunit interactions occurred. These results are consistent with interactions observed in a crystal structure of eukaryotic RNA polymerase II and support a common archaeal/eukaryal RNA polymerase architecture. We further propose that subunit E" is not an integral member of archaeal RNA polymerases. Finally, we discuss the relative accuracy of the various methods that have been used to predict protein-protein interactions in RNA polymerase.

• Toulokhonov I, Artsimovitch I, Landick R
• Allosteric control of RNA polymerase by a site that contacts nascent RNA hairpins.
• Science. 2001; 292: 730-3
• Display abstract

• DNA, RNA, and regulatory molecules control gene expression through interactions with RNA polymerase (RNAP). We show that a short alpha helix at the tip of the flaplike domain that covers the RNA exit channel of RNAP contacts a nascent RNA stem-loop structure (hairpin) that inhibits transcription, and that this flap-tip helix is required for activity of the regulatory protein NusA. Protein-RNA cross-linking, molecular modeling, and effects of alterations in RNAP and RNA all suggest that a tripartite interaction of RNAP, NusA, and the hairpin inhibits nucleotide addition in the active site, which is located 65 angstroms away. These findings favor an allosteric model for regulation of transcript elongation.

• Andreeva OI et al.
• [Interaction of HIV-1 reverse transcriptase and bacteriophage T7 RNA polymerase with NTP phosphonate analogs and inorganic pyrophosphate].
• Mol Biol (Mosk). 2001; 35: 844-56
• Display abstract

• We have examined the interaction of human immunodeficiency virus reverse transcriptase (HIV RT) and T7 RNA polymerase (T7 RNAP) with modified nucleoside triphosphates and inorganic pyrophosphate (PPi) analogs containing nonhydrolyzable bisphosphonate groups. We have synthesized a number of derivatives of bisphosphonic acid having different aromatic and nonaromatic side substituents, as well as the NTP derivatives whose incorporation into the growing nucleotide chain during the polymerization reaction results in formation of bisphosphonates as leaving groups. The competitive character of inhibition of both enzymes has been revealed for all the compounds under study, and the inhibition constants have been estimated. One of PPi analogs containing a bulky aromatic substituent is characterized by similar inhibition constants for both T7 RNAP and RT. The universal character of this inhibitor can serve as evidence for a similar structure of the NPT-binding sites in the two polymerases. It has been shown that nonsubstituted methylenebisphosphate is a better leaving group than that containing additional methyl and hydroxyl groups. The NTP analogs are very weak inhibitors of T7 RNAP, whereas HIV-1 RT is more sensitive to this type of compounds. On the basis of the X-ray crystallographic data on the T7 RNAP complex with a template and NTP, we have modeled the binding of some derivatives of bisphosphonic acid in the active center of the enzyme. The peculiarities observed in the model correlate well with the experimental data on inhibition.

• Diamond JM, Turner DH, Mathews DH
• Thermodynamics of three-way multibranch loops in RNA.
• Biochemistry. 2001; 40: 6971-81
• Display abstract

• RNA multibranch loops (junctions) are loops from which three or more helices exit. They are nearly ubiquitous in RNA secondary structures determined by comparative sequence analysis. In this study, systems in which two strands combine to form three-way junctions were used to measure the stabilities of RNA multibranch loops by UV optical melting and isothermal titration calorimetry (ITC). These data were used to calculate the free energy increment for initiation of a three-way junction on the basis of a nearest neighbor model for secondary structure stability. Imino proton NMR spectra were also measured for two systems and are consistent with the hypothesized helical structures. Incorporation of the experimental data into the mfold and RNA structure computer programs has contributed to an improvement in prediction of RNA secondary structure from sequence.

• Song H, Kang C
• Sequence-specific termination by T7 RNA polymerase requires formation of paused conformation prior to the point of RNA release.
• Genes Cells. 2001; 6: 291-301
• Display abstract

• BACKGROUND: The sequence-specific, hairpin-independent termination signal for the bacteriophage RNA polymerases in Escherichia coli rrnB t1 terminator consists of two modules. The upstream module includes the conserved sequence and the downstream one is U-rich. RESULTS: Elongation complexes of T7 RNA polymerase paused 2 bp before reaching the termination site at a 500 microM concentration of NTP. At 5-50 microM NTP, however, they paused and terminated there or resumed elongation beyond the termination site. Only at higher concentrations of NTP (500 microM), the pause complex proceeded slowly to and became incompetent at the termination site. At 4 bp or more before the termination site, the unprotected single-stranded region of transcription bubble shrank at the trailing edge to 4-5 bp from approximately 10 bp, resulting from duplex formation of the conserved sequence. The pause and bubble collapse were not observed with an inactive mutant of the termination signal. CONCLUSION: Sequence-specific termination requires the slow elongation mode of paused conformation, working only at high concentrations of NTP for a few bp prior to the RNA release site. The collapse of bubble that was observed several base pairs before the termination site and/or the resulting duplex might subsequently lead to the paused conformation of T7 elongation complexes.

• Brieba LG, Sousa R
• The T7 RNA polymerase intercalating hairpin is important for promoter opening during initiation but not for RNA displacement or transcription bubble stability during elongation.
• Biochemistry. 2001; 40: 3882-90
• Display abstract

• The recently described crystal structures of a T7RNAP-promoter complex and an initial transcription complex reveal a beta-hairpin which inserts between the template and nontemplate strands of the promoter [Cheetham, G. M., et al. (1999) Nature 399, 80; Cheetham, G. M., et al. (1999) Science 286, 2305]. A stacking interaction between the exposed DNA bases and a valine at the tip of this hairpin may be especially important for stabilizing the opened promoter during initiation. It has been suggested that this hairpin may also be important for holding the transcription bubble open during transcript elongation, and a proposed model for how the RNA exits the transcription complex implies that this hairpin may also help displace the RNA from the template strand. To test these hypotheses, we have characterized both point and deletion mutants of this element. We find that these mutants exhibit reduced activity on linear, double-stranded templates but not on supercoiled or partially single-stranded templates. Probing of promoter-polymerase complexes, initial transcription complexes, and elongation complexes with KMnO(4) and a single-strand specific endonuclease reveals that the mutants have greatly reduced promoter unwinding activity during initiation. However, the structure and stability of the transcription bubble during elongation are not altered in the mutant enzymes, and RNA displacement activity is also normal. Thus, the T7RNAP intercalating hairpin is important, though not essential, for stabilizing the opened promoter during initiation, but is not important for RNA displacement or for transcription bubble structure or stability during elongation.

• Jiang M, Rong M, Martin C, McAllister WT
• Interrupting the template strand of the T7 promoter facilitates translocation of the DNA during initiation, reducing transcript slippage and the release of abortive products.
• J Mol Biol. 2001; 310: 509-22
• Display abstract

• We have explored the effects of a variety of structural and sequence changes in the initiation region of the phage T7 promoter on promoter function. At promoters in which the template strand (T strand) is intact, initiation is directed a minimal distance of 5 nt downstream from the binding region. Although the sequence of the DNA surrounding the start site is not critical for correct initiation, it is important for melting of the promoter and stabilization of the initiation complex. At promoters in which the integrity of T strand is interrupted by nicks or gaps between -5 and -2 the enzyme continues to initiate predominately at +1. However, under these conditions there is a decrease in the release of abortive products of 8-10 nt, a decrease in the synthesis of poly(G) products (which arise by slippage of the nascent transcript), and a defect in displacement of the RNA. We propose that unlinking the binding and initiation regions of the T strand changes the manner in which this strand is retained in the abortive complex, reducing or eliminating the need to pack or "scrunch" the strand into the complex during initiation and lowering a thermodynamic barrier to its translocation.

• Cramer P, Bushnell DA, Kornberg RD
• Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.
• Science. 2001; 292: 1863-76
• Display abstract

• Structures of a 10-subunit yeast RNA polymerase II have been derived from two crystal forms at 2.8 and 3.1 angstrom resolution. Comparison of the structures reveals a division of the polymerase into four mobile modules, including a clamp, shown previously to swing over the active center. In the 2.8 angstrom structure, the clamp is in an open state, allowing entry of straight promoter DNA for the initiation of transcription. Three loops extending from the clamp may play roles in RNA unwinding and DNA rewinding during transcription. A 2.8 angstrom difference Fourier map reveals two metal ions at the active site, one persistently bound and the other possibly exchangeable during RNA synthesis. The results also provide evidence for RNA exit in the vicinity of the carboxyl-terminal repeat domain, coupling synthesis to RNA processing by enzymes bound to this domain.

• Butcher SJ, Grimes JM, Makeyev EV, Bamford DH, Stuart DI
• A mechanism for initiating RNA-dependent RNA polymerization.
• Nature. 2001; 410: 235-40
• Display abstract

• In most RNA viruses, genome replication and transcription are catalysed by a viral RNA-dependent RNA polymerase. Double-stranded RNA viruses perform these operations in a capsid (the polymerase complex), using an enzyme that can read both single- and double-stranded RNA. Structures have been solved for such viral capsids, but they do not resolve the polymerase subunits in any detail. Here we show that the 2 A resolution X-ray structure of the active polymerase subunit from the double-stranded RNA bacteriophage straight phi6 is highly similar to that of the polymerase of hepatitis C virus, providing an evolutionary link between double-stranded RNA viruses and flaviviruses. By crystal soaking and co-crystallization, we determined a number of other structures, including complexes with oligonucleotide and/or nucleoside triphosphates (NTPs), that suggest a mechanism by which the incoming double-stranded RNA is opened up to feed the template through to the active site, while the substrates enter by another route. The template strand initially overshoots, locking into a specificity pocket, and then, in the presence of cognate NTPs, reverses to form the initiation complex; this process engages two NTPs, one of which acts with the carboxy-terminal domain of the protein to prime the reaction. Our results provide a working model for the initiation of replication and transcription.

• Ujvari A, Martin CT
• Evidence for DNA bending at the T7 RNA polymerase promoter.
• J Mol Biol. 2000; 295: 1173-84
• Display abstract

• Phage T7 RNA polymerase is the only DNA-dependent RNA polymerase for which we have a high-resolution structure of the promoter-bound complex. Recent studies with the more complex RNA polymerases have suggested a role for DNA wrapping in the initiation of transcription. Here, circular permutation gel retardation assays provide evidence that the polymerase does indeed bend its promoter DNA. A complementary set of experiments employing differential phasing from an array of phased A-tracts provides further evidence for both intrinsic and polymerase-induced bends in the T7 RNA polymerase promoter DNA. The bend in the complex is predicted to be about 40-60 degrees and to be centered around positions -2 to +1, at the start site for transcription, while the intrinsic bend is much smaller (about 10 degrees ). These results, viewed in the light of a recent crystal structure for the complex, suggest a mechanism by which binding leads directly to bending. Bending at the start site would then facilitate the melting necessary to initiate transcription.

• Temiakov D, Mentesana PE, Ma K, Mustaev A, Borukhov S, McAllister WT
• The specificity loop of T7 RNA polymerase interacts first with the promoter and then with the elongating transcript, suggesting a mechanism for promoter clearance.
• Proc Natl Acad Sci U S A. 2000; 97: 14109-14
• Display abstract

• During the early stages of transcription, T7 RNA polymerase forms an unstable initiation complex that synthesizes and releases transcripts 2-8 nt in length before disengaging from the promoter and isomerizing to a stable elongation complex. In this study, we used RNA small middle dotprotein and RNA small middle dotDNA crosslinking methods to probe the location of newly synthesized RNA in halted elongation complexes. The results indicate that the RNA in an elongation complex remains in an RNA small middle dotDNA hybrid for about 8 nt from the site of nucleotide addition and emerges to the surface of the enzyme about 12 nt from the addition site. Strikingly, as the transcript leaves its hybrid with the template, the crosslinks it forms with the RNA polymerase involve a portion of a hairpin loop (the specificity loop) that makes specific contacts with the binding region of the promoter during initiation. This observation suggests that the specificity loop may have a dual role in transcription, binding first to the promoter and subsequently interacting with the RNA product. It seems likely that association of the nascent RNA with the specificity loop facilitates disengagement from the promoter and is an important part of the process that leads to a stable elongation complex.

• Mentesana PE, Chin-Bow ST, Sousa R, McAllister WT
• Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability.
• J Mol Biol. 2000; 302: 1049-62
• Display abstract

• We have constructed a series of plasmid templates that allow T7 RNA polymerase (RNAP) to be halted at defined intervals downstream from its promoter in a preserved sequence context. While transcription complexes halted at +3 to +6 are highly unstable, complexes halted at +10 to +14 dissociate very slowly and gradually lose their capacity to extend transcripts. Complexes halted at +18 and beyond dissociate more readily, but the stability of the these complexes is enhanced significantly in the presence of the next incoming nucleotide. Unexpectedly, the stability of complexes halted at +14 and beyond was found to be lower on supercoiled templates than on linear templates. To explore this further, we used synthetic DNA templates in which the nature of the non-template (NT) strand was varied. Whereas initiation complexes are less stable in the presence of a complementary NT strand, elongation complexes are more stable in the presence of a complementary NT strand, and the presence of a non-complementary NT strand (a mismatched bubble) results in even greater stability. The results suggest that the NT strand plays an important role in displacing the nascent RNA, allowing its interaction with an RNA product binding site in the RNAP. The NT strand may also contribute to stabilization by interacting directly with the enzyme. A mutant RNAP that has a deletion in the flexible "thumb" domain responds to changes in template topology in a manner that is similar to that of the wild-type (WT) enzyme, but halted complexes formed by the mutant enzyme are particularly dependent upon the presence of the NT strand for stability. In contrast, an N-terminal RNAP mutant that has a decreased capacity to bind single-stranded RNA forms halted complexes with much lower levels of stability than the WT enzyme, and these complexes are not stabilized by the presence of the NT strand. The distinct responses of the mutant RNAPs to changes in template structure indicate that the N-terminal and thumb domains have quite different functions in stabilizing the transcription complex.

• Cramer P et al.
• Architecture of RNA polymerase II and implications for the transcription mechanism.
• Science. 2000; 288: 640-9
• Display abstract

• A backbone model of a 10-subunit yeast RNA polymerase II has been derived from x-ray diffraction data extending to 3 angstroms resolution. All 10 subunits exhibit a high degree of identity with the corresponding human proteins, and 9 of the 10 subunits are conserved among the three eukaryotic RNA polymerases I, II, and III. Notable features of the model include a pair of jaws, formed by subunits Rpb1, Rpb5, and Rpb9, that appear to grip DNA downstream of the active center. A clamp on the DNA nearer the active center, formed by Rpb1, Rpb2, and Rpb6, may be locked in the closed position by RNA, accounting for the great stability of transcribing complexes. A pore in the protein complex beneath the active center may allow entry of substrates for polymerization and exit of the transcript during proofreading and passage through pause sites in the DNA.

• Conaway JW, Conaway RC
• Structural biology. Light at the end of the channel.
• Science. 2000; 288: 632-3

• Beletskii A, Grigoriev A, Joyce S, Bhagwat AS
• Mutations induced by bacteriophage T7 RNA polymerase and their effects on the composition of the T7 genome.
• J Mol Biol. 2000; 300: 1057-65
• Display abstract

• We show here that transcription by the bacteriophage T7 RNA polymerase increases the deamination of cytosine bases in the non-transcribed strand to uracil, causing C to T mutations in that strand. Under optimal conditions, the mutation frequency increases about fivefold over background, and is similar to that seen with the Escherichia coli RNA polymerase. Further, we found that a mutant T7 RNA polymerase with a slower rate of elongation caused more cytosine deaminations than its wild-type parent. These results suggest that promoting cytosine deamination in the non-transcribed strand is a general property of transcription in E. coli and is dependent on the length of time the transcription bubble stays open during elongation. To see if transcription-induced mutations have influenced the evolution of bacteriophage T7, we analyzed its genome for a bias in base composition. Our analysis showed a significant excess of thymine over cytosine bases in the highly transcribed regions of the genome. Moreover, the average value of this bias correlated well with the levels of transcription of different genomic regions. Our results indicate that transcription-induced mutations have altered the composition of bacteriophage T7 genome and suggest that this may be a significant force in genome evolution.

• Yoo J, Kang C
• Bacteriophage SP6 RNA polymerase mutants with altered termination efficiency and elongation processivity.
• Biomol Eng. 2000; 16: 191-7
• Display abstract

• An Escherichia coli strain containing two plasmids was developed for in vivo isolation of the phage SP6 RNA polymerase mutants. It was developed to isolate mutants with increased proficiency of termination at the SP6 terminator and/or with reduced elongation processivity. Mutations were randomly introduced into an N-terminal third of the polymerase gene that was placed under a lac promoter in one plasmid. In the other plasmid, a promoter-lacking lacZ gene modified for reduced translation efficiency was placed downstream of a tandem pair of the SP6 terminator located downstream of an SP6 promoter-chloramphenicol acetyltransferase gene. Termination-up mutants were selected in vivo as they rendered LacZ activity level lower than the wild-type, without reducing chloramphenicol resistance substantially. Three such mutants (M15L, M15S, and D117G) were purified, and their termination efficiencies were measured in vitro at two different intrinsic termination signals in the E. coli rrnB terminator t1 that are different in requiring RNA hairpin formation. All three mutations enhanced termination efficiencies in vitro at the SP6 terminator and the upstream signal of rrnB t1, but reduced the efficiency at the downstream signal of it. All the mutations reduced elongation processivity, as the mutants produced much less amounts of large transcripts (2.1 kb) than the wild-type but the similar amounts of small transcripts (up to 670 nt). Thus, the mutations, all reducing elongation processivity of the polymerase, exhibited opposite effects on the two types of intrinsic termination signals, suggesting that the two mechanisms involve different interactions with the phage RNA polymerase.

• Kamzolova SG, Ivanova NN, Sorokin AA, Kamzalov SS
• Promoter specificity of Escherichia coli rpoB403 mutant RNA polymerase.
• Dokl Biol Sci. 2000; 372: 333-5

• Mackereth CD, Arrowsmith CH, Edwards AM, McIntosh LP
• Zinc-bundle structure of the essential RNA polymerase subunit RPB10 from Methanobacterium thermoautotrophicum.
• Proc Natl Acad Sci U S A. 2000; 97: 6316-21
• Display abstract

• The RNA polymerase subunit RPB10 displays a high level of conservation across archaea and eukarya and is required for cell viability in yeast. Structure determination of this RNA polymerase subunit from Methanobacterium thermoautotrophicum reveals a topology, which we term a zinc-bundle, consisting of three alpha-helices stabilized by a zinc ion. The metal ion is bound within an atypical CX(2)CX(n)CC sequence motif and serves to bridge an N-terminal loop with helix 3. This represents an example of two adjacent zinc-binding Cys residues within an alpha-helix conformation. Conserved surface features of RPB10 include discrete regions of neutral, acidic, and basic residues, the latter being located around the zinc-binding site. One or more of these regions may contribute to the role of this subunit as a scaffold protein within the polymerase holoenzyme.

• Zhang G, Campbell EA, Minakhin L, Richter C, Severinov K, Darst SA
• Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 A resolution.
• Cell. 1999; 98: 811-24
• Display abstract

• The X-ray crystal structure of Thermus aquaticus core RNA polymerase reveals a "crab claw"-shaped molecule with a 27 A wide internal channel. Located on the back wall of the channel is a Mg2+ ion required for catalytic activity, which is chelated by an absolutely conserved motif from all bacterial and eukaryotic cellular RNA polymerases. The structure places key functional sites, defined by mutational and cross-linking analysis, on the inner walls of the channel in close proximity to the active center Mg2+. Further out from the catalytic center, structural features are found that may be involved in maintaining the melted transcription bubble, clamping onto the RNA product and/or DNA template to assure processivity, and delivering nucleotide substrates to the active center.

• Asturias FJ, Kornberg RD
• Protein crystallization on lipid layers and structure determination of the RNA polymerase II transcription initiation complex.
• J Biol Chem. 1999; 274: 6813-6

• Chirinos M, Hernandez F, Palacian E
• Transcription of DNA templates associated with histone (H3 x H4)(2) tetramers.
• Arch Biochem Biophys. 1999; 370: 222-30
• Display abstract

• To investigate the in vitro transcription by bacteriophage T7 RNA polymerase of oligonucleosomes lacking histone H2A x H2B dimers, templates were assembled from histone (H3 x H4)(2) tetramers with and without the complementary amount of H2A x H2B dimers and two different DNA species: pGEMEX-1, devoid of nucleosome positioning sequences, and T7-207-18, which contains downstream from the promoter 18 tandem repeats of a 207-bp positioning sequence. Assembly with core histone octamers affects pGEMEX-1 transcription mainly at the initiation level, while T7-207-18 is almost exclusively inhibited at the level of elongation. With both DNA templates and under different salt conditions, RNA synthesis is much more efficient on oligonucleosomes containing only (H3 x H4)(2) tetramers than on those with whole histone octamers. Under conditions promoting a low transcription rate, it is unambiguously shown with pGEMEX-1 that the block to initiation due to the presence of core histone octamers is substantially removed when (H3 x H4)(2) is substituted for the whole octamer. With T7-207-18, under assay conditions allowing transcription of the whole coding region of the naked DNA, analysis of the transcription products indicates that RNA elongation on the template containing (H3 x H4)(2) tetramers takes place as easily as on free DNA, in contrast with the significant inhibition observed in the presence of whole histone octamers.

• Nudler E
• Transcription elongation: structural basis and mechanisms.
• J Mol Biol. 1999; 288: 1-12
• Display abstract

• A ternary complex composed of RNA polymerase (RNAP), DNA template, and RNA transcript is the central intermediate in the transcription cycle responsible for the elongation of the RNA chain. Although the basic biochemistry of RNAP functioning is well understood, little is known about the underlying structural determinants. The absence of high- resolution structural data has hampered our understanding of RNAP mechanism. However, recent work suggests a structure-function model of the ternary elongation complex, if not at a defined structural level, then at least as a conceptual view, such that key components of RNAP are defined operationally on the basis of compelling biochemical, protein chemical, and genetic data. The model has important implications for mechanisms of transcription elongation and also for initiation and termination.

• Lipniacki T
• Chemically driven traveling waves in DNA.
• Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics. 1999; 60: 7253-61
• Display abstract

• The nonlinear mechanical model constructed in a previous paper [Nuovo Cimento D 20, 833 (1998)] is developed in order to study the dynamics of the DNA double helix. It is assumed that the hydrophobic interaction between subsequent base pairs may be influenced by a RNA polymerase. The Lagrangian, constructed on the basis of "geometrical" properties of the DNA molecule, depends on time and contains first and second derivatives of the twist angle. The energy dissipation term is added to the dynamical equations resulting from the Lagrange formalism. It is proved that the system has pulse-like solitary wave solutions for which the dissipated energy is balanced by the energy pumped by the advancing RNA polymerase. The physical interpretation of our solution is the local untwisting of the DNA molecule during transcription of messenger RNA.

• Chen CJ, Liu ZJ, Rose JP, Wang BC
• Low-salt crystallization of T7 RNA polymerase: a first step towards the transcription bubble complex.
• Acta Crystallogr D Biol Crystallogr. 1999; 55: 1188-92
• Display abstract

• DNA-dependent RNA polymerase is the key enzyme responsible for the biosynthesis of RNA, a process known as transcription. This process, which decodes the genetic information from DNA, is one of the most significant events in a biological system. The crystallization of both native and a chimeric T7/T3 RNAP using high salt conditions has been reported previously but these conditions proved unsuitable for DNA-RNAP complex formation since at high salt concentrations the DNA binding affinity to RNAP is reduced. A search for low-salt crystallization conditions has yielded new low-salt crystals of native T7-RNAP, a chimeric T7-RNAP (T7/T3 RNAP) which contains the T3 promoter recognition sequence, and a T7-RNAP containing an N-terminal histidine tag. The crystals, which are better suited for DNA-RNAP complex formation, belong to space group P3121 with a = 136, c = 156 A, contain a single molecule per asymmetric unit and diffract to 2.7 A resolution. Packing analysis shows that the new low-salt crystals have packing contacts similar to those observed in the high-salt T7-RNAP crystals reported previously. The diffraction anisotropicity observed in crystals of T7 RNAP is explained in term of crystal packing.

• Cheetham GM, Jeruzalmi D, Steitz TA
• Structural basis for initiation of transcription from an RNA polymerase-promoter complex.
• Nature. 1999; 399: 80-3
• Display abstract

• Although the single-polypeptide-chain RNA polymerase from bacteriophage T7 (T7RNAP), like other RNA polymerases, uses the same mechanism of polymerization as the DNA polymerases, it can also recognize a specific promoter sequence, initiate new RNA chains from a single nucleotide, abortively cycle the synthesis of short transcripts, be regulated by a transcription inhibitor, and terminate transcription. As T7RNAP is homologous to the Pol I family of DNA polymerases, the differences between the structure of T7RNAP complexed to substrates and that of the corresponding DNA polymerase complex provides a structural basis for understanding many of these functional differences. T7RNAP initiates RNA synthesis at promoter sequences that are conserved from positions -17 to +6 relative to the start site of transcription. The crystal structure at 2.4 A resolution of T7RNAP complexed with a 17-base-pair promoter shows that the four base pairs closest to the catalytic active site have melted to form a transcription bubble. The T7 promoter sequence is recognized by interactions in the major groove between an antiparallel beta-loop and bases. The amino-terminal domain is involved in promoter recognition and DNA melting. We have also used homology modelling of the priming and incoming nucleoside triphosphates from the T7 DNA-polymerase ternary complex structure to explain the specificity of T7RNAP for ribonucleotides, its ability to initiate from a single nucleotide, and the abortive cycling at the initiation of transcription.

• Arnaud-Barbe N, Cheynet-Sauvion V, Oriol G, Mandrand B, Mallet F
• Transcription of RNA templates by T7 RNA polymerase.
• Nucleic Acids Res. 1998; 26: 3550-4
• Display abstract

• Although highly specialized, T7 RNA polymerase seems to possess a large range of DNA- and RNA-dependent properties. To study such flexibility, we determined the ability of T7 RNA polymerase to transcribe chimeric DNA-RNA and RNA templates following initiation at a double stranded DNA promoter. We have found that T7 RNA polymerase is able to initiate on RNA templates, was processive, and was able to use templates containing RNA-RNA duplexes under standard transcription conditions. Implications of remnants of such RNA-dependent activities for T7 DNA-dependent polymerase are discussed.

• Jeruzalmi D, Steitz TA
• Structure of T7 RNA polymerase complexed to the transcriptional inhibitor T7 lysozyme.
• EMBO J. 1998; 17: 4101-13
• Display abstract

• The T7 RNA polymerase-T7 lysozyme complex regulates phage gene expression during infection of Escherichia coli. The 2.8 A crystal structure of the complex reveals that lysozyme binds at a site remote from the polymerase active site, suggesting an indirect mechanism of inhibition. Comparison of the T7 RNA polymerase structure with that of the homologous pol I family of DNA polymerases reveals identities in the catalytic site but also differences specific to RNA polymerase function. The structure of T7 RNA polymerase presented here differs significantly from a previously published structure. Sequence similarities between phage RNA polymerases and those from mitochondria and chloroplasts, when interpreted in the context of our revised model of T7 RNA polymerase, suggest a conserved fold.

• Zhang G, Darst SA
• Structure of the Escherichia coli RNA polymerase alpha subunit amino-terminal domain.
• Science. 1998; 281: 262-6
• Display abstract

• The 2.5 angstrom resolution x-ray crystal structure of the Escherichia coli RNA polymerase (RNAP) alpha subunit amino-terminal domain (alphaNTD), which is necessary and sufficient to dimerize and assemble the other RNAP subunits into a transcriptionally active enzyme and contains all of the sequence elements conserved among eukaryotic alpha homologs, has been determined. The alphaNTD monomer comprises two distinct, flexibly linked domains, only one of which participates in the dimer interface. In the alphaNTD dimer, a pair of helices from one monomer interact with the cognate helices of the other to form an extensive hydrophobic core. All of the determinants for interactions with the other RNAP subunits lie on one face of the alphaNTD dimer. Sequence alignments, combined with secondary-structure predictions, support proposals that a heterodimer of the eukaryotic RNAP subunits related to Saccharomyces cerevisiae Rpb3 and Rpb11 plays the role of the alphaNTD dimer in prokaryotic RNAP.

• Nudler E, Gusarov I, Avetissova E, Kozlov M, Goldfarb A
• Spatial organization of transcription elongation complex in Escherichia coli.
• Science. 1998; 281: 424-8
• Display abstract

• During RNA synthesis in the ternary elongation complex, RNA polymerase enzyme holds nucleic acids in three contiguous sites: the double-stranded DNA-binding site (DBS) ahead of the transcription bubble, the RNA-DNA heteroduplex-binding site (HBS), and the RNA-binding site (RBS) upstream of HBS. Photochemical cross-linking allowed mapping of the DNA and RNA contacts to specific positions on the amino acid sequence. Unexpectedly, the same protein regions were found to participate in both DBS and RBS. Thus, DNA entry and RNA exit occur close together in the RNA polymerase molecule, suggesting that the three sites constitute a single unit. The results explain how RNA in the integrated unit RBS-HBS-DBS may stabilize the ternary complex, whereas a hairpin in RNA result in its dissociation.

• Brautigam CA, Steitz TA
• Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes.
• Curr Opin Struct Biol. 1998; 8: 54-63
• Display abstract

• New levels in the understanding of DNA replication have been achieved from recent crystal structure determinations of several DNA polymerases and their substrate complexes. The structure of an alpha family DNA polymerase from bacteriophage RB69 shows some similarities, but also considerable differences in structure and organization from the pol I family DNA polymerases. Also, the functions of three polymerase domains and their conserved residues have been clarified by studying structures of pol I family DNA polymerases complexed to their substrates. These structures also confirm that an identical two-metal ion catalytic mechanism proposed previously is used by both the nonhomologous pol I and pol beta family DNA polymerases.

• Lopez PJ, Guillerez J, Sousa R, Dreyfus M
• On the mechanism of inhibition of phage T7 RNA polymerase by lac repressor.
• J Mol Biol. 1998; 276: 861-75
• Display abstract

• We study here the effect on phage T7 RNA polymerase activity of lac repressor bound downstream of the T7 promoter. When repressor binds in vitro at an operator centered at +13 or +15 with respect to transcription start, it does not prevent initiation, though the transcript yield is reduced. However, the processivity of the polymerase is depressed and transcript extension is blocked at positions +4 and +6, respectively. These results indicate that repressor and polymerase do not simply exclude each other from the promoter. Rather, they would come into steric conflict and compete for establishment or retention of interactions with the same segment of DNA, without this leading to the immediate displacement of either polymerase or repressor. The resulting destabilization of the transcription complex would depress both initiation rate and enzyme processivity. In contrast to the above results, little reduction in runoff transcription is observed when operator is centered at +47. The decreased sensitivity of polymerase to repressor bound at +47 versus +13 or +15 is likely to be due to the higher stability of the elongation complex during the transcription of downstream regions in comparison with the first transcribed nucleotides. We also show that under conditions of leaky repression and with operator centered at +13, a mutant T7 RNA polymerase showing normal promoter affinity but a slower elongation rate is more sensitive to repression than the wild-type enzyme, both in vitro and in vivo. In vitro, this higher sensitivity is largely due to a reduced ability of the mutant to overcome the elongation block at position +4. The parallel between the in vitro and in vivo data suggests that in vivo the repressor also does not prevent polymerase from binding to promoter, but interferes with subsequent steps in initiation and transcript extension, in this case presumably largely extension beyond +4.

• Erijman L, Clegg RM
• Reversible stalling of transcription elongation complexes by high pressure.
• Biophys J. 1998; 75: 453-62
• Display abstract

• We have investigated the effect of high hydrostatic pressure on the stability of RNA polymerase molecules during transcription. RNA polymerase molecules participating in stalled or active ternary transcribing complexes do not dissociate from the template DNA and nascent RNA at pressures up to 180 MPa. A lower limit for the free energy of stabilization of an elongating ternary complex relative to the quaternary structure of the free RNAP molecules is estimated to be 20 kcal/mol. The rate of elongation decreases at high pressure; transcription completely halts at sufficiently high pressure. The overall rate of elongation has an apparent activation volume (DeltaVdouble dagger) of 55-65 ml . mol-1 (at 35 degrees C). The pressure-stalled transcripts are stable and resume elongation at the prepressure rate upon decompression. The efficiency of termination decreases at the rho-independent terminator tR2 after the transcription reaction has been exposed to high pressure. This suggests that high pressure modifies the ternary complex such that termination is affected in a manner different from that of elongation. The solvent and temperature dependence of the pressure-induced inhibition show evidence for major conformational changes in the core polymerase enzyme during RNA synthesis. It is proposed that the inhibition of the elongation phase of the transcription reaction at elevated pressures is related to a reduction of the partial specific volume of the RNA polymerase molecule; under high pressure, the RNA polymerase molecule does not have the necessary structural flexibility required for the protein to translocate.

• Doublie S, Tabor S, Long AM, Richardson CC, Ellenberger T
• Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 A resolution.
• Nature. 1998; 391: 251-8
• Display abstract

• DNA polymerases change their specificity for nucleotide substrates with each catalytic cycle, while achieving error frequencies in the range of 10(-5) to 10(-6). Here we present a 2.2 A crystal structure of the replicative DNA polymerase from bacteriophage T7 complexed with a primer-template and a nucleoside triphosphate in the polymerase active site. The structure illustrates how nucleotides are selected in a template-directed manner, and provides a structural basis for a metal-assisted mechanism of phosphoryl transfer by a large group of related polymerases.

• Rodriguez-Monge L, Ouzounis CA, Kyrpides NC
• A ferredoxin-like domain in RNA polymerase 30/40-kDa subunits.
• Trends Biochem Sci. 1998; 23: 169-70

• Jia Y, Patel SS
• Kinetic mechanism of transcription initiation by bacteriophage T7 RNA polymerase.
• Biochemistry. 1997; 36: 4223-32
• Display abstract

• The kinetic mechanism of transcription initiation by bacteriophage T7 RNA polymerase was investigated using transient state kinetic methods. Transcription by bacteriophage T7 RNA polymerase occurs in three stages consisting of initiation, promoter clearance, and elongation. Abortive products, up to 6-8-mer, were synthesized during the initiation phase; the transition from initiation to elongation occurred between the synthesis of 6-8-mer and 11-12-mer, and the processive elongation phase began after the synthesis of 12-mer RNA. Our results show that the synthesis of elongation product from the phi 10 promoter is limited both by the efficiency of initiation and by the frequency at which the polymerase escapes the promoter. Studies with heparin trap suggest that the polymerase maintains contact with the promoter region during multiple turnovers of abortive RNA synthesis; thus, the polymerase does not completely dissociate from the promoter after each event of abortive RNA synthesis. The pre-steady-state kinetics of RNA synthesis indicate that initiation occurs at a rate constant (3.5 s(-1)) that is about 30 times faster than the steady-state rate constant of RNA synthesis (0.1 s(-1)). The steady-state rate constant of RNA synthesis is limited largely by the cycling of the RNA polymerase, whereas initiation is limited by the formation of pppGpG, the first RNA product. We show that the synthesis of pppGpG is not limited by steps associated with GTP binding, DNA binding, or the melting of the promoter DNA. Instead, the kinetic results indicate that initiation at the phi10 promoter is limited either by the first phosphodiester bond formation step or more likely by a conformational change prior to pppGpG formation. Such a conformational change could play a role in proper alignment of the initiating and elongating NTPs for efficient phosphodiester bond formation and in maintaining the fidelity of RNA synthesis.

• Arnaud N, Cheynet V, Oriol G, Mandrand B, Mallet F
• Construction and expression of a modular gene encoding bacteriophage T7 RNA polymerase.
• Gene. 1997; 199: 149-56
• Display abstract

• A modular gene that encodes T7 RNA polymerase (T7 RNAP) and consists of cassettes delimited by unique restriction sites was constructed. The modular and wild-type genes of T7 RNAP were cloned into a vector designed to express His-tagged proteins. The modular and wild-type genes provided the same level of protein expression (i.e., T7 RNAP represented up to 30% of the total protein in Escherichia coli strain BL21). Purification of both proteins by immobilized metal ion affinity chromatography (IMAC) resulted in similar yields (700-800 microg of enzyme per 20 ml of culture) and purity (>95%) as indicated by Coomassie blue staining, Western blotting and the absence of detectable contaminating nuclease activities. Both proteins exhibited identical efficiency in transcription assays, and their specific activities (about 200 U/microg) were close to that of a commercial T7 RNAP preparation. The modular gene provides a useful tool for cassette directed mutagenesis of T7 RNAP.

• Tunitskaia VL, Memelova LV, Kostiuk DA, Gudima SO, Kochetkov SN
• [Regularities in utilization of deoxyribonucleoside triphosphates by mutant forms of bacteriophage T7 RNA polymerase].
• Mol Biol (Mosk). 1997; 31: 353-8

• Sastry SS, Ross BM
• A direct real-time spectroscopic investigation of the mechanism of open complex formation by T7 RNA polymerase.
• Biochemistry. 1996; 35: 15715-25
• Display abstract

• Initiation of transcription occurs through a series of steps starting with the binding of RNA polymerase to a promoter DNA and formation of a closed complex. The closed complexes, then isomerize to open complexes. In the open complexes a portion of the promoter DNA is unwound. Using fluorescence spectroscopy, we have investigated in real-time the mechanism of unwinding of promoter DNA during the transition from closed to open complexes of T7 RNA polymerase. We synthesized DNA templates containing the fluorescent base analog 2-aminopurine in place of adenine at specific positions in a T7 RNA polymerase promoter. We located the 2-aminopurine residues in the presumed melting domain of the promoter at -1, -4, and at -6. The fluorescence of 2-aminopurine increases when the DNA goes from a double-stranded form to a single-stranded form. By spectroscopically monitoring the increase in fluorescence of 2-aminopurine in DNA-T7 RNA polymerase complexes, we obtained kinetic and thermodynamic information for DNA unwinding. In the presence of the initiating nucleotide GTP, conformational transitions in the polymerase-promoter complex leading to strand opening were slower than in its absence. The rate of base pair disruption at -1, -6, and at -4 was also slower in the presence of GTP than in its absence. At 37 degrees C, base pair disruption occurred first at -1 followed by -6 and finally at -4. Open complex formation was temperature-sensitive. Temperature effects at -1, -6, and at -4 were consistent with this order of base pair disruption. The apparent activation energies (Ea) for base pair disruption around -1 and -6 were 14 kcal mol-1 and 50 kcal mol-1, respectively, also suggesting this order of base pair disruption. Transcription initiation assays using G-ladder synthesis revealed that initiation rates were almost the same on all three templates containing the modified base. Unlike strand opening, we did not observe lag times for G-ladder synthesis. We suggest that facile base pair disruption at -1 is sufficient for transcription initiation. Based on these data, it is proposed that the polymerase makes contacts at or near -1 and -6 resulting in untwisting of these base pairs thus creating at least two base pair disruption events at -1 and at -6, which are followed by bidirectional propagation to -4.

• Choi DJ, Roth RB, Liu T, Geacintov NE, Scicchitano DA
• Incorrect base insertion and prematurely terminated transcripts during T7 RNA polymerase transcription elongation past benzo[a]pyrenediol epoxide-modified DNA.
• J Mol Biol. 1996; 264: 213-9
• Display abstract

• DNA replication and transcription are affected adversely by the presence of bulky adducts that are generated by the covalent binding of a variety of metabolically activated environmental pollutants to cellular DNA. When these lesions are not cleared by cellular repair enzymes prior to replication, mutations and ultimately tumor initiation can occur. Transcription and DNA repair appear to be intimately connected, since certain adducts are more efficiently removed from the transcribed strands of active loci than from non-transcribed strands and other quiescent domains in the genome. The mechanism by which RNA polymerases deal with bulky adducts during DNA transcription is therefore of great interest. The availability of site-specifically modified and stereochemically defined oligodeoxyribonucleotides derived from the covalent reaction of 7r, 8t-dihydroxy-9, 10t-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) with guanine residues prompted us to study the efficiencies of transcription past these lesions using bacteriophage T7 RNA polymerase. We show here that T7 RNA polymerase can bypass such lesions in a DNA template, providing that a cytosine residue is incorporated opposite anti-BPDE-modified guanine. However, when an incorrect base (most frequently a purine) is inserted opposite the modified site, the RNA polymerase stalls, and the complex dissociates, resulting in a truncated transcript. The ability of the T7 RNA polymerase to discriminate between a correct and an incorrect inserted base and, accordingly, to continue or terminate transcription, might constitute an important mechanism that ensures the fidelity of transcription past a modified base present on the transcribed strand of the DNA template.

• Minnick DT, Astatke M, Joyce CM, Kunkel TA
• A thumb subdomain mutant of the large fragment of Escherichia coli DNA polymerase I with reduced DNA binding affinity, processivity, and frameshift fidelity.
• J Biol Chem. 1996; 271: 24954-61
• Display abstract

• In Klenow fragment DNA polymerase, a flexible 50-amino acid subdomain at the tip of the thumb which includes two alpha helices has been suggested to interact with the duplex template-primer (Beese, L.S., Derbyshire, V. and Steitz, T.A. (1993) Science 260, 352-355). The present study investigates the properties of Klenow polymerase containing a 24-amino acid deletion (residues 590-613) that removes a portion of the tip of the thumb. The mutant polymerase has relatively normal dNTP binding and catalytic rate. However, its DNA binding affinity is reduced by more than 100-fold relative to the intact polymerase and its ability to conduct processive synthesis is also reduced. Although the mutant polymerase has relatively normal base substitution fidelity, it has strongly reduced frameshift fidelity, being especially error-prone for single nucleotide addition errors in homopolymeric runs. The addition error rate increases as the length of the reiterated sequence increases, indicative of errors initiated by template-primer strand slippage. These observations suggest a role for the tip of the thumb of Klenow polymerase in determining DNA binding, processivity and frameshift fidelity, perhaps by tracking the minor groove of the duplex DNA. The results are discussed in light of remarkably similar observations with T7 DNA polymerase in the presence or absence of thioredoxin, an accessory subunit that affects these same properties.

• Yu FL, Cahill JM, Lipinski LJ, Dipple A
• Effect of aflatoxin B1-8,9-epoxide-DNA adducts on transcription of a supF gene fragment.
• Cancer Lett. 1996; 109: 77-83
• Display abstract

• A linearized template, obtained from the vector pGEM-3Zf(+) containing a supF gene fragment, was treated with aflatoxin B1-8,9-epoxide (AFB1 epoxide) and transcription in vitro was then studied. The template functions of both strands of the supF gene were similarly inhibited as shown by transcription with both T7 and SP6 RNA polymerases. This inhibition was dose-dependent and affected the elongation step more extensively than the initiation step. Gel electrophoretic analysis of RNA formed by T7 RNA polymerase indicated that template treated with different AFB1 epoxide doses yielded the same three major truncated RNA fragments. Sequence analysis showed that these major sites of RNA truncation occurred in the vicinity of adjacent guanine residues in the template.

• Baklanov MM, Golikova LN, Malygin EG
• [Effect of the structure of the prepromotor region on transcription using phage T7 RNA polymerase].
• Mol Biol (Mosk). 1996; 30: 449-53

• Zhou W, Reines D, Doetsch PW
• T7 RNA polymerase bypass of large gaps on the template strand reveals a critical role of the nontemplate strand in elongation.
• Cell. 1995; 82: 577-85
• Display abstract

• We show that T7 RNA polymerase can efficiently transcribe DNA containing gaps from one to five bases in the template strand. Surprisingly, broken template strands missing up to 24 bases can still be transcribed, although at reduced efficiency. The resulting transcripts contain the full template sequence with the RNA deleted for the gapped region missing on the template strand. These findings indicate that the end of a downstream template strand can be brought into the polymerase and transcribed as if it were a part of an intact polynucleotide chain by utilizing the unpaired nontemplate strand. This, as well as transcription of an intact template strand, relies heavily upon the non-template strand, suggesting that a duplex DNA-binding site on the leading edge of RNA polymerase is required for RNA chain elongation on DNA templates. This work contributes substantially to the emerging picture that the nontemplate strand is an important element of the transcription elongation complex.

• Oubridge C, Ito N, Teo CH, Fearnley I, Nagai K
• Crystallisation of RNA-protein complexes. II. The application of protein engineering for crystallisation of the U1A protein-RNA complex.
• J Mol Biol. 1995; 249: 409-23
• Display abstract

• The hairpin is one of the most commonly found structural motifs of RNA and is often a binding site for proteins. Crystallisation of U1A spliceosomal protein bound to a RNA hairpin, its natural binding site on U1snRNA, is described. RNA oligonucleotides were synthesised either chemically or by in vitro transcription using T7 RNA polymerase and purified to homogeneity by gel electrophoresis. Crystallisation trials with the wild-type protein sequence and RNA hairpins containing various stem sequences and overhanging nucleotides only resulted in a cubic crystal form which diffracted to 7-8 A resolution. A new crystal form was grown by using a protein variant containing mutations of two surface residues. The N-terminal sequence of the protein was also varied to reduce heterogeneity which was detected by protein mass spectrometry. A further crystallisation search using the double mutant protein and varying the RNA hairpins resulted in crystals diffracting to beyond 1.7 A. The methods and strategy described in this paper may be applicable to crystallisation of other RNA-protein complexes.

• Fleischmann RD et al.
• Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.
• Science. 1995; 269: 496-512
• Display abstract

• An approach for genome analysis based on sequencing and assembly of unselected pieces of DNA from the whole chromosome has been applied to obtain the complete nucleotide sequence (1,830,137 base pairs) of the genome from the bacterium Haemophilus influenzae Rd. This approach eliminates the need for initial mapping efforts and is therefore applicable to the vast array of microbial species for which genome maps are unavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase accession number L42023) represents the only complete genome sequence from a free-living organism.

• Liu J, Zhou W, Doetsch PW
• RNA polymerase bypass at sites of dihydrouracil: implications for transcriptional mutagenesis.
• Mol Cell Biol. 1995; 15: 6729-35
• Display abstract

• Dihydrouracil (DHU) is a major base damage product formed from cytosine following exposure of DNA to ionizing radiation under anoxic conditions. To gain insight into the DNA lesion structural requirements for RNA polymerase arrest or bypass at various DNA damages located on the transcribed strand during elongation, DHU was placed onto promoter-containing DNA templates 20 nucleotides downstream from the transcription start site. In vitro, single-round transcription experiments carried out with SP6 and T7 RNA polymerases revealed that following a brief pause at the DHU site, both enzymes efficiently bypass this lesion with subsequent rapid generation of full-length runoff transcripts. Direct sequence analysis of these transcripts indicated that both RNA polymerases insert primarily adenine opposite to the DHU site, resulting in a G-to-A transition mutation in the lesion bypass product. Such bypass and insertion events at DHU sites (or other types of DNA damages), if they occur in vivo, have a number of important implications for both the repair of such lesions and the DNA damage-induced production of mutant proteins at the level of transcription (transcriptional mutagenesis).

• Kostiuk DA, Dragan SM, Rechinskii VO, Tunitskaia VL, Chernov BK, Kochetkov SN
• [A mutant form of bacteriophage T7 RNA polymerase capable of catalyzing RNA and DNA synthesis].
• Dokl Akad Nauk. 1995; 341: 824-7

• Schick C, Martin CT
• Tests of a model of specific contacts in T7 RNA polymerase-promoter interactions.
• Biochemistry. 1995; 34: 666-72
• Display abstract

• The T7, T3, and SP6 RNA polymerases represent a highly homologous family of enzymes that recognize similarly homologous promoter DNA sequences. Despite these similarities, the enzymes are highly specific for their respective promoters. Studies of mutant RNA polymerases have linked a specific amino acid residue in the protein to recognition of bases at positions -11 and -10 in the promoter [Raskin, C. A., et al. (1992) J. Mol. Biol. 228, 506-515]. In kinetic analyses of transcription from synthetic promoters containing base-analog substitutions, we have recently shown that at positions -11 and -10 of the T3 promoter, T3 RNA polymerase recognizes functional groups along the nontemplate strand wall of the major groove [Schick, C., & Martin, C. T. (1993) Biochemistry 32, 4275-4780]. We now extend these studies to the homologous region of the T7 promoter. The results confirm extrapolations from the T3 system and show that T7 RNA polymerase recognizes corresponding functional groups at positions -11 and -10 of the T7 promoter. The results are consistent with a direct readout model for recognition of these bases [Raskin, C. A., et al. (1992) J. Mol. Biol., 228, 506-515], in which the 6-carbonyl and 7-imino groups of the nontemplate guanine at position -11 and the 6-amino group of the nontemplate adenine at position -10 of the T7 promoter are directly involved in binding. The results further support an overall model for promoter recognition in which the enzyme binds to one face of the duplex DNA in this upstream region of the promoter.

• Meyer-Almes FJ, Heumann H, Porschke D
• The structure of the RNA polymerase-promoter complex. DNA-bending-angle by quantitative electrooptics.
• J Mol Biol. 1994; 236: 1-6
• Display abstract

• The complex formed between RNA polymerase holoenzyme from Escherichia coli and the strong promoter A1 from the phage T7 has been characterized by measurements of the electric dichroism. The dichroism decay time constant of a promoter DNA fragment with 126 bp increases upon binding of the polymerase, but the increase is less than expected for simple addition of the components at the known binding site. Our results demonstrate a protein-induced decrease of the hydrodynamic DNA dimensions, which is not a consistent with an increased flexibility, but indicates bending of the double helix with a relatively narrow distribution of bending angles. We have characterized the degree of DNA bending by bead model simulations and used, in addition to our present experimental data, the available information on the overall size and shape of the RNA polymerase, together with the location of the DNA bending center at the starting point of RNA synthesis. We conclude that the bending angle is 45 degrees (+/- 5 degrees).

• Maslak M, Martin CT
• Effects of solution conditions on the steady-state kinetics of initiation of transcription by T7 RNA polymerase.
• Biochemistry. 1994; 33: 6918-24
• Display abstract

• The T7 family of DNA-dependent RNA polymerases presents an ideal model system for the study of fundamental aspects of transcription. The small size of the promoter allows a variety of studies based on simple steady-state kinetics in the synthesis of a five-base runoff transcript. This assay can be used to characterize the effects on the initiation of transcription of simple modifications to potential protein or DNA specificity contacts. In the current work, in vitro conditions for this assay have been identified which optimize the apparent Km for the interaction between the enzyme and the promoter DNA. The addition to the reaction mixture of 0.05% Tween-20 and the substitution of 10 mM NaCl by 100 mM potassium glutamate not only improves the quality of the kinetic assays but also decreases Km by about an order of magnitude (strengthening the interaction between polymerase and its promoter). As observed for DNA binding in other systems, the parameter Km increases substantially with increasing [NaCl], but the salt dependence is shifted to higher concentrations as a function of [KGlu]. Thermal denaturation of the protein, monitored by circular dichroism spectroscopy, confirms the effects of salt and supports a model in which Cl- and other anions compete for phosphate binding sites on the protein. Finally, while Km is highly dependent on [NaCl], the measured kcat is relatively insensitive to salt. These data indicate that the parameters Km and kcat reflect changes respectively in promoter binding and in a rate-limiting step or steps leading to the initiation of transcription.

• Joyce CM, Steitz TA
• Function and structure relationships in DNA polymerases.
• Annu Rev Biochem. 1994; 63: 777-822

• Mikita T, Beardsley GP
• Effects of arabinosylcytosine-substituted DNA on DNA/RNA hybrid stability and transcription by T7 RNA polymerase.
• Biochemistry. 1994; 33: 9195-208
• Display abstract

• Cytosine arabinoside (araC) is a potent antileukemic agent which interferes with DNA replication both as a dNTP competitive inhibitor as well as after its misincorporation into DNA. We previously developed a chemical methodology for the synthesis of DNA oligomers containing araC which allowed us to study its site specific effects on duplex stability and chemical reactivity [Beardsley, G. P., Mikita, T., Klaus, M., & Nussbaum, A. (1988) Nucleic Acids Res. 16, 9165], as well as its effects on DNA ligase and DNA polymerase activity [Mikita, T., & Beardsley, G. P. (1988) Biochemistry 27, 4698]. The DNA polymerase studies, in addition to other observations, showed that araC in DNA templates could have an inhibitory effect on polymerase bypass. As a template lesion, there exists the potential for interference with other aspects of DNA metabolism, such as transcription. We have characterized a DNA/RNA hybrid containing an araC-G base pair, comparing thermal stability, chemical cleavage rates, and duplex gel mobility to an identically sequenced DNA duplex. We find that the A-form DNA/RNA hybrid and the B-form DNA duplex are nearly identical in the extent their thermal stability is affected by an araC-G(dG) base pair. Substitutions of araC for dC were made at various positions in a series of DNA duplex substrates containing a T7 RNA polymerase promoter with variable length coding strands. These were used to probe the effect of araC on promoter recognition, initiation, and elongation by T7 RNA polymerase in vitro. Substitutions in the central promoter region had no observable effect on RNA polymerase binding, initiation rate, or transcriptional output. Coding strand substitutions defined an area of high sensitivity in the initiation region where miss-starts, primer slippage, and an inability to escape from abortive cycling occur depending on the position substituted. Substitutions after position 10 had little effect on transcription output. These highly variable, position dependent effects indicate a narrow window of vulnerability where transcription output is severely reduced (approximately 100-fold) by a subtle DNA lesion that has little or no consequence when situated elsewhere in these small coding units.

• Bonner G, Lafer EM, Sousa R
• The thumb subdomain of T7 RNA polymerase functions to stabilize the ternary complex during processive transcription.
• J Biol Chem. 1994; 269: 25129-36
• Display abstract

• To examine the function of the thumb subdomain in bacteriophage T7 RNA polymerase we constructed a set of deletion mutants within this subdomain. These mutants exhibited reduced processivity during the processive, but not the abortive, stage of transcription. Reduced processivity was found to be due primarily to an increase in the processive ternary complex dissociation rate (destabilization of the processive ternary complex). The destabilization of the ternary complex does not appear to be due to a decrease in the affinity of the polymerase for the nascent RNA. These observations support the proposal that the thumb subdomain functions to stabilize the processive ternary complex during the processive (but not the abortive) stage of transcription, probably by wrapping around the template to prevent polymerase dissociation.

• Steitz TA, Smerdon SJ, Jager J, Joyce CM
• A unified polymerase mechanism for nonhomologous DNA and RNA polymerases.
• Science. 1994; 266: 2022-5

• Holm L, Sander C
• Searching protein structure databases has come of age.
• Proteins. 1994; 19: 165-73
• Display abstract

• The number of protein structures known in atomic detail has increased from one in 1960 (Kendrew, J.C., Strandberg, B.E., Hart, R.G., Davies, D.R., Phillips, D.C., Shore, V.C. Nature (London) 185:422-427, 1960) to more than 1000 in 1994. The rate at which new structures are being published exceeds one a day as a result of recent advances in protein engineering, crystallography, and spectroscopy. More and more frequently, a newly determined structure is similar in fold to a known one, even when no sequence similarity is detectable. A new generation of computer algorithms has now been developed that allows routine comparison of a protein structure with the database of all known structures. Such structure database searches are already used daily and they are beginning to rival sequence database searches as a tool for discovering biologically interesting relationships.

• Weiss RA
• How RNA makes DNA.
• Science. 1994; 264: 1954-5

• Daube SS, von Hippel PH
• RNA displacement pathways during transcription from synthetic RNA-DNA bubble duplexes.
• Biochemistry. 1994; 33: 340-7
• Display abstract

• Previously [Daube, S.S., & von Hippel, P.H. (1992) Science 258, 1320] we have shown that functional transcription elongation complexes can be formed by adding ribonucleotide triphosphates, Mg2+, and either Escherichia coli or T7 RNA polymerase to synthetic RNA-DNA bubble-duplex constructs. Here these observations are extended to show that the RNA transcripts synthesized from these bubble-duplex constructs are properly displaced from the DNA template during transcription. Some details of the displacement process differ between the polymerases tested. Thus the transcript is fully and processively displaced in the course of T7 polymerase-catalyzed synthesis from the bubble-duplex constructs, while the presence of a large excess of an RNA (or DNA) oligomer complementary to the DNA template sequence within the "permanent" DNA bubble is required to attain complete displacement of the nascent RNA from the construct during synthesis with the core E. coli enzyme. In addition, a correlation is shown between proper RNA displacement and the achievement of full-length transcript synthesis. We conclude that both the T7 polymerase and the E. coli core enzyme actively displace the nascent transcript during elongation and that the requirement for an RNA trap with the E. coli enzyme reflects its slower rate of synthesis. This suggests that these experiments may provide insight into the relative rates of transcript elongation and secondary structure formation within the nascent RNA in elongation and termination. By use of the RNA oligomer trap methodology, multiple rounds of transcript synthesis should be achievable on these bubble-duplex constructs with any polymerase.

• Sastry SS, Spielmann HP, Hoang QS, Phillips AM, Sancar A, Hearst JE
• Laser-induced protein-DNA cross-links via psoralen furanside monoadducts.
• Biochemistry. 1993; 32: 5526-38
• Display abstract

• We have developed a technique for cross-linking DNA binding proteins to DNA using psoralen furanside monoadducts as photoaffinity probes and a continuous-wave argon ion laser (366 nm) as a light source. Several DNA binding proteins (T7 RNA polymerase, UvrB, single-stranded DNA binding protein of Escherichia coli, T4 gp32, and RecA of E. coli) are shown to cross-link to single-stranded psoralen monoadducted DNA oligos differing in length and sequence. Increasing fluences of laser light on a fixed ratio of DNA/protein resulted in an increase in the yield of cross-links. Titration experiments were carried out to measure the apparent cross-linking constant (KappXL) for T7 RNA polymerase or UvrB to a monoadducted 24 mer DNA. The estimated values for the apparent cross-linking constant were in the range of (2-3) x 10(-7) M for both T7 RNA polymerase and UvrB. The efficiency of cross-linking was investigated as a function of the length of adducted DNA and also as a fraction of the total noncovalent binding of proteins of psoralenated DNAs. The results showed that in the cases of T7 RNA polymerase and UvrB cross-linking was more efficient with short oligos (8 and 19 mers) as compared to longer oligos (50 mer). A tryptic peptide of T7 RNA polymerase that was conjugated to a psoralen furanside monoadducted 12 mer DNA was isolated by high-performance liquid chromatography. Mass spectrometry and amino acid composition of this peptide revealed that it originated from a region between residues 558 and 608 of the primary structure of T7 RNA polymerase. Two other peptides cross-linked to oligos were also purified. Repeated attempts to perform Edman sequencing of the peptide-DNA conjugates failed. Overall evidence indicates that photo-cross-linking of furanside monoadducts occurred at multiple sites on the proteins. We have shown that T7 RNA polymerase molecules in a ternary complex arrested at the furanside monoadduct can be cross-linked to the DNA templates with laser light. Evidence suggests that the arrested polymerase molecules existed in multiple conformations on the DNA template. This method of transcriptional cross-linking offers a new method for preparing highly stable elongation complexes for further studies.

• Skoog JU, Maher LJ 3rd
• Repression of bacteriophage promoters by DNA and RNA oligonucleotides.
• Nucleic Acids Res. 1993; 21: 2131-8
• Display abstract

• We are interested in creating artificial gene repressors based on duplex DNA recognition by nucleic acids rather than polypeptides. An in vitro model system involving repression of bacteriophage T7 RNA polymerase initiation has been employed to demonstrate that certain DNA oligonucleotides can repress transcription by site-specific triple-helix formation at two kinds of homopurine operator sequences [Maher, L. J., III, (1992) Biochemistry 31, 7587-7594]. Recognition in the purine motif is based on antiparallel oligonucleotide binding (G.G.C and T.A.T triplets). Recognition in the pyrimidine motif is based on parallel oligonucleotide binding (C+.G.C and T.A.T base triplets). Using this system, we report that the concentration-dependence of repression by DNA oligonucleotides provides triple-helix inhibition constant (Ki) estimates of approximately 2 x 10(-7) M for both purine motif and pyrimidine motif DNA complexes. RNA oligonucleotides are shown to repress promoters overlapping pyrimidine motif operators (Ki = 6 x 10(-7) M), but not purine motif operators. Although competent to hybridize to complementary single strands, RNA oligonucleotides fail to bind the purine motif operator. Partial substitution of deoxyribose residues tends to rescue repressor activity by RNA oligonucleotides in the purine motif. These results suggest prospects for, and constraints on, natural and artificial RNA-based repressors.

• van der Vliet PC, Verrijzer CP
• Bending of DNA by transcription factors.
• Bioessays. 1993; 15: 25-32
• Display abstract

• An increasing number of transcription factors both from prokaryotic and eukaryotic sources are found to bend the DNA upon binding to their recognition site. Bending can easily be detected by the anomalous electrophoretic behaviour of the DNA-protein complex or by increased cyclization of DNA fragments containing the protein-induced bend. Induction of DNA bending by transcription factors could regulate transcription in various ways. Bending may bring distantly bound transcription factors closer together by facilitating DNA-looping or it could mediate the interaction between transcription factors and the general transcription machinery by formation of large nucleoprotein structures in which the DNA is wrapped around the protein complex. Alternatively, the energy stored in a protein-induced bend could be used to favour formation of an open transcription complex or to dissociate the RNA polymerase in the transition from initiation to elongation. Modification of the bend angles and bending centers, caused by homodimerization or heterodimerization of transcription factors, may well turn out to be an important way to enlarge the range of interactions required for regulation of gene expression.

• Lee SS, Kang C
• Two base pairs at -9 and -8 distinguish between the bacteriophage T7 and SP6 promoters.
• J Biol Chem. 1993; 268: 19299-304
• Display abstract

• Bacteriophage T7 and SP6 RNA polymerases and their promoters share a high degree of their primary structure homology, but each polymerase exclusively recognizes its own promoter sequence. To reveal the molecular basis of this specificity, 4 base pairs at positions -12, -10, -9, and -8 of the T7 promoter were substituted individually and multiply by SP6 promoter-specific base pairs, and 3 base pairs at -10, -9, and -8 of the SP6 promoter were replaced by T7 promoter-specific base pairs. Promoter activities of 28 sequences were measured in vitro with T7 and SP6 polymerases separately under optimal conditions at 6 mM MgCl2. Single and double substitutions at -12 and -10 do not significantly affect the T7 promoter activity, although they are almost exclusively conserved among T7 genomic promoters. Changes at -10 of SP6 promoter hardly affect the activity. However, any T7 variants that contain either or both changes at -9 and -8 show greatly reduced activity. Interestingly, the double substitution at -9 and -8 yields significant SP6 promoter activities and virtually no T7 promoter activity. Furthermore, the SP6 promoter variants with both T7-specific -9C and -8T show good T7 promoter activities, although they still show some SP6 promoter activities. However, under high salt conditions (either 20 mM MgCl2 or 100 mM NaCl plus 6 mM MgCl2), they show only slight SP6 promoter activity. No other SP6 variants show any T7 promoter activity. All these results indicate that the 2 base pairs at -9 and -8 of both the T7 and SP6 promoters are the primary (if not the only) determinants of specificity and that the hierarchy of importance of positions for promoter activity is -8, -9 > > -10 > -12. Also, a phylogenic relationship among the T3, T7, K11, and SP6 promoters is suggested based on dissimilarities in their sequences from -12 to -8.

• Taylor DR, Mathews MB
• Transcription by SP6 RNA polymerase exhibits an ATP dependence that is influenced by promoter topology.
• Nucleic Acids Res. 1993; 21: 1927-33
• Display abstract

• Transcription of linearized DNA templates by SP6 RNA polymerase requires a higher concentration of ATP than of the other three nucleotides. This requirement is not shared by T7 RNA polymerase. The ATP requirement is partially relieved when the SP6 template is supercoiled but not when it is relaxed circular DNA. The effect of supercoiling is eliminated by replacement of the A.T rich sequence downstream from the SP6 promoter with a G.C rich sequence. Examination of the reaction products indicates that the ATP dependence of transcription from a linear template is not due to an ATPase activity or to the premature termination of transcription at low ATP concentration. These data suggest that the initiation of transcription by SP6 RNA polymerase requires partial denaturation of the template in the promoter-proximal region, and that this requirement can be satisfied by negative supercoiling or by increasing the ATP concentration. ATP also reduces, but does not eliminate, the abortive transcription that leads to the production of short, prematurely terminated transcripts by SP6 polymerase from supercoiled templates.

• Tunitskaia VL et al.
• [Study of the form of bacteriophage T7 RNA polymerase containing point substitutions in the region of functionally important amino acid residues].
• Mol Biol (Mosk). 1993; 27: 92-102
• Display abstract

• A study was carried out on the influence of point mutations of the functional amino acid residues on the secondary and ternary structure of bacteriophage T7 RNA polymerase as well as on the activity of the enzyme. A change in residue 631 is accompanied by significant changes in secondary structure (alpha-->beta transition). The substitution Lys-172 Leu changes both the secondary and ternary structures whereas the deletion of residues 172-173 does not lead to such changes. Changes in residues 631-632 do not affect the ability of the enzyme to bind the promoter and/or the synthesis of a full-length transcript but disturbs phosphodiester bond formation.

• Dunaway M, Ostrander EA
• Local domains of supercoiling activate a eukaryotic promoter in vivo.
• Nature. 1993; 361: 746-8
• Display abstract

• Experiments correlating template topology with transcriptional activity suggest that DNA topology plays a role in eukaryotic gene expression. Linear templates transfected into cultured cells produce far fewer transcripts than do circular transcription templates, and no transcripts can be detected from linear templates injected into Xenopus oocytes. Further, when transcriptionally active circular templates in Xenopus oocytes are linearized by injection of a restriction enzyme, transcription dramatically decreases. Here we show that transcription by phage T7 RNA polymerase from a divergent promoter can partially replace the requirement for circular Xenopus ribosomal RNA transcription templates in Xenopus oocytes. Supercoiled domains can apparently be generated on short pieces of DNA having no known sequences that result in association with the nuclear architecture, suggesting that localized, transient domains of supercoiling fulfil the minimum topological needs for Xenopus rRNA transcription in vivo.

• Rechinsky VO, Tunitskaya VL, Dragan SM, Kostyuk DA, Kochetkov SN
• Tyr-571 is involved in the T7 RNA polymerase binding to its promoter.
• FEBS Lett. 1993; 320: 9-12
• Display abstract

• The in vitro studies of three T7 RNA polymerase point mutants suggest that substitutions of Ala and Thr for Pro-563 and of Ser for Tyr-571 have little effect on the enzyme catalytic competence, but result in its inability to utilize the promoter. Both P563A and P563T mutants retain the promoter-binding ability, whereas the promoter affinity of the Y571S mutant drops drastically.

• Bonner G, Patra D, Lafer EM, Sousa R
• Mutations in T7 RNA polymerase that support the proposal for a common polymerase active site structure.
• EMBO J. 1992; 11: 3767-75
• Display abstract

• In order to test the proposal that most nucleotide polymerases share a common active site structure and folding topology, we have generated 22 mutations of residues within motifs A, B and C of T7 RNA polymerase (RNAP). Characterization of these T7 RNAP mutants showed the following: (i) most of the mutations resulted in moderate to drastic reductions in T7 RNAP transcriptional activity supporting the idea that motifs A, B and C identify part of the polymerase active site; (ii) the degree of conservation of an amino acid within these motifs correlated with the degree to which mutation of that amino acid reduced transcriptional activity, supporting the predictive ability of this alignment in identifying the most functionally critical residues; (iii) a comparison of DNAP I and T7 RNAP mutants revealed similarities (as well as differences) between corresponding mutant phenotypes; (iv) the Klenow fragment structure is shown to provide a reasonable basis for interpretation of the differential effects of mutating different amino acids within motifs A, B and C in T7 RNAP. These observations support the proposal that these polymerase active sites have similar three-dimensional structures.

• Golub EI, Ward DC, Radding CM
• Joints formed by RecA protein from oligonucleotides and duplex DNA block initiation and elongation of transcription.
• Nucleic Acids Res. 1992; 20: 3121-5
• Display abstract

• In the presence of the non-hydrolyzable analog of ATP, ATP gamma S, RecA protein can polymerize on an oligodeoxy-ribonucleotide to form a stable oligonucleoprotein filament that can find its homologous sequence in double-stranded DNA. The homologous joint formed by the oligonucleotide and duplex DNA is stable only if RecA protein is not removed. Such a nucleoprotein joint, covering a part or all of the promoter region of T3 or T7 phage RNA polymerase, blocked transcription directed by those polymerases. The same kind of joint, located downstream of the RNA polymerase promoter, also inhibited elongation of transcription and caused accumulation of truncated transcripts. These observations suggest that RecA protein can be used to shut off transcription from any promoter of known sequence.

• Sastry SS, Hearst JE
• Studies on the interaction of T7 RNA polymerase with a DNA template containing a site-specifically placed psoralen cross-link. I. Characterization of elongation complexes.
• J Mol Biol. 1991; 221: 1091-110
• Display abstract

• A 66 base-pair (bp) DNA template carrying a site-specifically placed psoralen cross-link downstream from a phage T7 promoter was constructed. This template can support transcription by T7 RNA polymerase. Transcription was blocked specifically at the psoralen cross-link. We studied the characteristics of elongation complexes, formed in this manner, by enzymatic and chemical footprinting and by a nitrocellulose filter-binding assay. The DNase I footprint of the elongation complex was quantified on a residue by residue basis. It was found that T7 RNA polymerase made the strongest contacts in the central region of the footprint whereas the leading and lagging edges of the polymerase were weakly bound to the DNA. Reducing the NaCl concentration in the transcription reaction resulted in the visualization of two T7 RNA polymerase molecules bound to the same template. A leading polymerase molecule, arrested at the psoralen cross-link, showed a much smaller DNase I footprint than a lagging polymerase molecule that was bound upstream. This upstream polymerase molecule occupied approximately one-half of the promoter region and therefore did not achieve complete promoter clearance. These experiments suggest that complete promoter clearance is required for a gross conformational change in the polymerase, consisting of a contraction in the size of its footprint to occur. DNase I footprinting also revealed that an elongation complex arrested at a psoralen cross-link undergoes several subtle changes in structure in a time-dependent manner and therefore can be considered to be in a state of dynamic flux. Methylation protection showed that some G residues in the top (non-coding) strand are protected against attack by dimethylsulfate, whereas the G residues on the bottom (coding) strand appear not to be protected from reaction with dimethylsulfate. We probed the transcribing complexes for single-stranded regions with T7 gene 3 endonuclease. From the pattern of sensitivity to T7 gene 3 endonuclease on the template strand, we conclude that the RNA-DNA hybrid in the elongation complex is about 7 bp. A nitrocellulose filter-binding assay showed that the elongation complex, consisting of a 36 (+1) nucleotide RNA, the 66 bp DNA template and the T7 RNA polymerase was stable for at least 30 minutes at high salt concentrations. Heparin caused the quantitative release of 36 (+1) RNA nucleotides within 30 seconds, but the DNA was not simultaneously released from the elongation complex under these conditions.

• Delarue M, Poch O, Tordo N, Moras D, Argos P
• An attempt to unify the structure of polymerases.
• Protein Eng. 1990; 3: 461-7
• Display abstract

• With the great availability of sequences from RNA- and DNA-dependent RNA and DNA polymerases, it has become possible to delineate a few highly conserved regions for various polymerase types. In this work a DNA polymerase sequence from bacteriophage SPO2 was found to be homologous to the polymerase domain of the Klenow fragment of polymerase I from Escherichia coli, which is known to be closely related to those from Staphylococcus pneumoniae, Thermus aquaticus and bacteriophages T7 and T5. The alignment of the SPO2 polymerase with the other five sequences considerably narrowed the conserved motifs in these proteins. Three of the motifs matched reasonably all the conserved motifs of another DNA polymerase type, characterized by human polymerase alpha. It is also possible to find these three motifs in monomeric DNA-dependent RNA polymerases and two of them in DNA polymerase beta and DNA terminal transferases. These latter two motifs also matched two of the four motifs recently identified in 84 RNA-dependent polymerases. From the known tertiary architecture of the Klenow fragment of E. coli pol I, a spatial arrangement can be implied for these motifs. In addition, numerous biochemical experiments suggesting a role for the motifs in a common function (dNTP binding) also support these inferences. This speculative hypothesis, attempting to unify polymerase structure at least locally, if not globally, under the pol I fold, should provide a useful model to direct mutagenesis experiments to probe template and substrate specificity in polymerases.

• Martin CT, Muller DK, Coleman JE
• Processivity in early stages of transcription by T7 RNA polymerase.
• Biochemistry. 1988; 27: 3966-74
• Display abstract

• Immediately following initiation of transcription, T7 RNA polymerase enters a phase in which dissociation of the enzyme-DNA-RNA ternary complex significantly competes with elongation, a process referred to in the Escherichia coli enzyme as abortive cycling [Carpousis, A.J., & Gralla, J.D. (1980) Biochemistry 19, 3245-3253]. Characterization of this process in the T7 RNA polymerase system under various reaction conditions and on templates with differing message sequences reveals that conversion to a highly processive ternary complex occurs after incorporation of eight bases and that the relative competition between dissociation and elongation up to this point is influenced by several different forces. In particular, the sequence dependence of abortive falloff suggests that dissociation is favored immediately following incorporation of UMP and is less likely following incorporation of GMP into the RNA message. Abortive cycling is unchanged in transcription from a synthetic oligonucleotide template which is double-stranded in the promoter region but single-stranded throughout the entire message region. This result proves that melting and reannealing of the DNA duplex in the coding region do not contribute to abortive cycling. Furthermore, weakening of promoter binding by an order of magnitude affects abortive cycling only slightly, suggesting that strong interactions with the promoter are not the major cause of abortive cycling. Kinetic analyses show that conversion to a highly processive ternary complex after the incorporation of eight bases may reflect a large decrease in the unimolecular rate of dissociation of the complex due to increased contacts between the nascent RNA and the DNA template and between RNA and enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

• Ruetsch N, Dennis D
• RNA polymerase. Limit cognate primer for initiation and stable ternary complex formation.
• J Biol Chem. 1987; 262: 1674-9
• Display abstract

• Various lengths of oligoribonucleotides corresponding to regions flanking the initiation site of the A1 promoter of the T7 delta D111 template were examined in order to determine their ability to function as primers of transcription for the DNA-dependent RNA polymerase from Escherichia coli. The oligoribonucleotides which functioned as primers were also examined as precursors for the formation of a stable ternary complex (enzyme X DNA template X oligoribonucleotide product) by systematically extending each primer with one or more specific cognate substrate nucleotide triphosphates. A stable ternary complex (resistant to a salt jump challenge) was formed whenever the primer oligoribonucleotide and augmenting nucleotide triphosphate(s) allowed the formation of the normal third phosphodiester bond of the transcript to occur on the enzyme surface. An oligoribonucleotide (a cognate having the correct base-pairing substituents) containing the preformed third phosphodiester bond does not function as a primer. For example the cognate oligoribonucleotide corresponding to the region flanking the A1 promoter of the T7 delta D111 is: formula; see text The limit primer is the oligoribonucleotide trimer AUC (+1...+3). Other acceptable primers are constructed by adding to this limit primer, cognate bases in the negative registry. The oligonucleotide containing one base added to this limit primer in the positive registry (e.g. AUCG) is completely inactive as a primer. We have also demonstrated these phenomena for the A2 and the A3 promoter of the T7 template.

• Smeekens SP, Romano LJ
• Promoter and nonspecific DNA binding by the T7 RNA polymerase.
• Nucleic Acids Res. 1986; 14: 2811-27
• Display abstract

• T7 RNA polymerase plays an important role in both the transcription and replication of bacteriophage T7. In this study we have used a nitrocellulose filter binding assay to examine the binding properties of the T7 RNA polymerase with T7 promoters cloned into plasmid DNAs. Promoter-specific binding was shown to be relatively insensitive to variations in the ionic strength of the incubation solution but dependent on the helical structure of the DNA. On the other hand, nonpromoter interior-site binding was independent of the superhelicity of the DNA but extremely sensitive to changes in the ionic strength. These results suggest that nonspecific binding results from ionic interactions between positively charged residues of the polymerase and the polyanionic backbone of the DNA, whereas promoter-specific binding is dependent upon base-specific contacts within the promoter sequence. A comparison between the transcriptional activity and binding strengths of the RNA polymerase to specific promoters indicates little correlation between these two properties. This suggests that differential promoter binding does not represent a major mechanism for regulating transcription in bacteriophage T7. Instead, factors which influence the efficiency or rate of formation of the polymerase-promoter open complex are found to have the major role in determining transcriptional levels in this system.

• Ollis DL, Brick P, Hamlin R, Xuong NG, Steitz TA
• Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP.
• Nature. 1985; 313: 762-6
• Display abstract

• The 3.3-A resolution crystal structure of the large proteolytic fragment of Escherichia coli DNA polymerase I complexed with deoxythymidine monophosphate consists of two domains, the smaller of which binds zinc-deoxythymidine monophosphate. The most striking feature of the larger domain is a deep crevice of the appropriate size and shape for binding double-stranded B-DNA. A flexible subdomain may allow the enzyme to surround completely the DNA substrate, thereby allowing processive nucleotide polymerization without enzyme dissociation.

• von Hippel PH, Bear DG, Morgan WD, McSwiggen JA
• Protein-nucleic acid interactions in transcription: a molecular analysis.
• Annu Rev Biochem. 1984; 53: 389-446

• Tsuji S, Imahori K, Nonomura Y
• The quaternary structure of DNA-dependent RNA polymerase.
• J Biochem. 1981; 89: 1903-12
• Display abstract

• The crystals of RNA polymerase of T. thermophilus were examined by electron microscopic observation of the negatively stained and sectioned materials. Three types of crystals were observed: ordered aggregates (Type I), cylindrical duplei (Type II), and plane (Type III) forms. It was deduced mainly from sectioned images that Type I crystal is a precursor or a premature form of Type II crystal. The III corresponds to the flattened layer of type II. In Type II crystal the enzyme molecules are arranged in an orderly two-dimensional lattice and thus we could analyze the molecular structure by optical filtering of the negatively stained images. On the basis of these results, a quaternary structure is proposed for RNA polymerase.

• Lescure B, Williamson V, Sentenac A
• Efficient and selective initiation by yeast RNA polymerase B in a dinucleotide-primed reaction.
• Nucleic Acids Res. 1981; 9: 31-45
• Display abstract

• Yeast RNA polymerase B catalyzes an efficient abortive initiation on double-stranded DNA templates using the appropriate combination of primer and substrate. The specificity of initiation was investigated using a recombinant plasmid (pJD14 DNA) containing the structural gene for yeast alcohol dehydrogenase I (ADHI). The combination of the dinucleotide UpA and UTP was 10 fold more efficient with pJD14 DNA than with the vector pBR322 DNA to direct the synthesis of the trinucleotide UpApU. Under these conditions, stable enzyme-DNA complexes were formed and could be retained on nitrocellulose filters. Using the UpA-primed system and a short pulse of RNA synthesis, transcription complexes were located on the yeast part of pJD14 DNA as evidenced by agarose gel electrophoresis. Southern hybridization of the pulsed RNA was restricted to a region, within the yeast DNA fragment, upstream to the initial region of the ADHI gene.

• McAllister WT, Morris C, Rosenberg AH, Studier FW
• Utilization of bacteriophage T7 late promoters in recombinant plasmids during infection.
• J Mol Biol. 1981; 153: 527-44

• Musielski H, Mann W, Laue R, Michel S
• Influence of dimethylsulfoxide on transcription by bacteriophage T3-induced RNA polymerase.
• Z Allg Mikrobiol. 1981; 21: 447-56
• Display abstract

• Dimethylsulfoxide (DMSO) up to 25% (v/v) does not cause irreversible alterations of T3 DNA at 42.5 degrees C as assayed by transcription with T3-specific RNA polymerase. The optimal temperature for the formation of polyanion-resistant ternary complexes of the enzyme, T3 DNA, and nascent RNA chains is lowered by 12.5 degrees C in the presence of 20% (v/v) DMSO. The same solvent concentration, however, decreased the temperature optimal for T3 RNA chain elongation by only 2.5 degrees C, indicating that DMSO preferably affects the initiation of T3 RNA synthesis. DMSO accelerates the loss of T3-specific RNA polymerase activity at 24.5 degrees C. Nevertheless, the speed with which the binary complexes between the phage RNA polymerase and DNA are inactivated by heat (42.5 degrees C) is not altered in presence of 20% (v/v) DMSO. The binding of T3-induced RNA polymerase to T3 DNA in polyanion-resistant ternary complexes is influenced by DMSO which makes the enzyme accessible to the inhibitory action of polyvinyl sulfate. Elongation of T3 RNA chains is slowed down by 20% (v/v) DMSO.

• Wever GH, Fischer H, Hinkle DC
• Bacteriophage T7 DNA replication in vitro. Electron micrographic analysis of T7 DNA synthesized with purified proteins.
• J Biol Chem. 1980; 255: 7965-72
• Display abstract

• Extensive replication of duplex T7 DNA is catalyzed in reactions contining T7 DNA polymerase, T7 gene 4 protein, and T7 RNA polymerase. When the product of this reaction is analyzed in the electron microscope, many eye form and Y form replication intermediates are observed. Replication in vitro is not initiated at a single region of the T7 genome. However, we tentatively conclude that initiation does occur preferentially at a few specific sites along the DNA, and that these sites may be near promoters at which the T7 RNA polymerase initiates transcription.

• McAllister WT, Carter AD
• Regulation of promoter selection by the bacteriophage T7 RNA polymerase in vitro.
• Nucleic Acids Res. 1980; 8: 4821-37
• Display abstract

• During bacteriophage T7 infection a phage-specified RNA polymerase transcribes the late phage genes in two temporal classes (class II and class III). In this report, we show that the purified phage polymerase discriminates between the class II and class III promoters in vitro as a function of variables that alter the stability of the DNA helix. These variables include ionic strength, temperature, and the presence of denaturing agents such as dimethyl sulfoxide. In general, initiation at the class II promoters is preferentially inhibited as helix stability is increased. Conditions required for the establishment of salt-resistant transcription complexes by the T7 RNA polymerase have been determined; the establishment of stable complexes at the class II promoters requires the synthesis of a longer nascent RNA transcript than does formation of such complexes at the class III promoters. A comparison of the nucleotide sequences of several class II and class III promoters suggests certain features that may be responsible for the different responses of these promoters to helix destabilization. The conservation of structural features that are peculiar to the class II or class III promoters indicates that these features are important in regulation of T7 transcription in vivo. Experiments which bear on the physiological significance of these features are discussed.

• Oakley JL, Coleman JE
• Structure of a promoter for T7 RNA polymerase.
• Proc Natl Acad Sci U S A. 1977; 74: 4266-70
• Display abstract

• We have determined the nucleotide sequence of a Hpa II restriction fragment of the phage T7 DNA containing a promoter for the phage-specified RNA polymerase. (Hpa II is a restriction endonuclease from Haemophilus parainfluenzae.) Mapping of the Hpa II restriction fragments on the T7 genome shows this promoter to be the second of tandem promoters separated by approximately 170 base pairs that begin transcription by the T7 RNA polymerase at approximately 15% of the genome. Features of the sequence involved in recognition by the T7 RNA polymerase are discussed and include the following region of hyphenated 2-fold symmetry (boxed regions are related through a 2-fold axis of symmetry at the center of the sequence shown). (See article). This sequence includes the initiation site, since the message transcribed from this fragment begins pppG-G-G-A. Combination of our results with work of others has permitted this fragment to be mapped at the junction of T7 genes 1 and 1.1. The RNA transcribed from this fragment begins within gene 1 and contains the RNase III cleavage site that lies between genes 1 and 1.1. This sequence is compared to other processing sites in T7 early message.

• Golomb M, Chamberlin M
• A preliminary map of the major transcription units read by T7 RNA polymerase on the T7 and T3 bacteriophage chromosomes.
• Proc Natl Acad Sci U S A. 1974; 71: 760-4
• Display abstract

• Transcription of T7 DNA by T7 RNA polymerase in vitro gives rise to six major size classes of RNAs comprising seven major T7 RNA species. These RNAs are all read from the r-strand of T7 DNA and are not derived from the early (leftmost on the conventional genetic map) region of the molecule. When artifically shortened T7 DNA templates are transcribed, four (I, II, IIIb, and VI) of the seven species are found to be truncated or deleted. This indicates that all are terminated near the right end of the T7 DNA molecule, probably at a common termination site near 98.5%. (Map positions are all given in terms of percentage of total length measured from the left end of the molecule.) Since the approximate lengths of the transcripts are known, the promotor sites for T7 RNA species I, II, IIIb, and VI are tentatively mapped at 56, 64, 83, and 97% on the T7 chromosome. Only a single major T3 RNA is transcribed by T7 RNA polymerase; analysis of transcripts directed by shortened T3 DNA templates indicates it is analogous to T7 RNA species IIIb. Hence the promotor and terminator sites for T3 species IIIb are tentatively mapped at 83 and 98.5%, respectively, on the T3 chromosome. The major transcripts read by T7 RNA polymerase from T3-T7 hybrid phage DNAs vary, depending on which regions of the T7 chromosome are present. This provides an alternative method of mapping the strong T7 promotor sites on the T7 chromosome.

• Singh UN
• Stringent coupling between transcription, translation, and degradation of messenger RNA in an inducible enzyme system: a theoretical analysis.
• Basic Life Sci. 1974; 3: 93-103

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