NORMAL GENOMIC

• Gries TJ, Kontur WS, Capp MW, Saecker RM, Record MT Jr
• One-step DNA melting in the RNA polymerase cleft opens the initiationbubble to form an unstable open complex.
• Proc Natl Acad Sci U S A. 2010; 107: 10418-23
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• Though opening of the start site (+1) region of promoter DNA is requiredfor transcription by RNA polymerase (RNAP), surprisingly little is knownabout how and when this occurs in the mechanism. Early events at thelambdaP(R) promoter load this region of duplex DNA into the active sitecleft of Escherichia coli RNAP, forming the closed,permanganate-unreactive intermediate I(1). Conversion to the subsequentintermediate I(2) overcomes a large enthalpic barrier. Is I(2) open? Herewe create a burst of I(2) by rapidly destabilizing open complexes (RP(o))with 1.1 M NaCl. Fast footprinting reveals that thymines at positions from-11 to +2 in I(2) are permanganate-reactive, demonstrating that RNAP opensthe entire initiation bubble in the cleft in a single step. Rates of decayof all observed thymine reactivities are the same as the I(2) to I(1)conversion rate determined by filter binding. In I(2), permanganatereactivity of the +1 thymine on the template (t) strand is the same as theRP(o) control, whereas nontemplate (nt) thymines are significantly lessreactive than in RP(o). We propose that: (i) the +1(t) thymine is in theactive site in I(2); (ii) conversion of I(2) to RP(o) repositions the ntstrand in the cleft; and (iii) movements of the nt strand are coupled tothe assembly and DNA binding of the downstream clamp and jaw that occursafter DNA opening and stabilizes RP(o). We hypothesize that unstable openintermediates at the lambdaP(R) promoter resemble the unstable,transcriptionally competent open complexes formed at ribosomal promoters.

• Feig M, Burton ZF
• RNA polymerase II flexibility during translocation from normal modeanalysis.
• Proteins. 2010; 78: 434-46
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• The structural dynamics in eukaryotic RNA polymerase II (RNAPII) isdescribed from computational normal mode analysis based on a series ofcrystal structures of pre- and post-translocated states with open andclosed trigger loops. Conserved modes are identified that involvetranslocation of the nucleic acid complex coupled to motions of theenzyme, in particular in the clamp and jaw domains of RNAPII. Acombination of these modes is hypothesized to be involved during activetranscription. The NMA modes indicate furthermore that downstream DNAtranslocation may occur separately from DNA:RNA hybrid translocation. Acomparison of the modes between different states of RNAPII suggests thatproductive translocation requires an open trigger loop and is inhibited bythe presence of an NTP in the active site. This conclusion is alsosupported by a comparison of the overall flexibility in terms of root meansquare fluctuations.

• Gangaraju VK, Prasad P, Srour A, Kagalwala MN, Bartholomew B
• Conformational changes associated with template commitment inATP-dependent chromatin remodeling by ISW2.
• Mol Cell. 2009; 35: 58-69
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• Distinct stages in ATP-dependent chromatin remodeling are found as ISW2,an ISWI-type complex, forms a stable and processive complex withnucleosomes upon hydrolysis of ATP. There are two conformational changesof the ISW2-nucleosome complex associated with binding and hydrolysis ofATP. The initial binding of ISW2 to extranucleosomal DNA, to the entrysite, and near the dyad axis of the nucleosome is enhanced by ATP binding,whereas subsequent ATP hydrolysis is required for template commitment andcauses ISW2 to expand its interactions with nucleosomal DNA to an entiregyre of the nucleosome and a short approximately 3-4 bp site on the othergyre. The histone-fold-like subunit Dpb4 associates with nucleosomal DNAapproximately 15 bp from the ATPase domain as part of this change and mayhelp to disrupt histone-DNA interactions. These additional contacts areindependent of the ATPase domain tracking along nucleosomal DNA and aremaintained as ISW2 moves nucleosomes on DNA.

• Svetlov V, Nudler E
• Jamming the ratchet of transcription.
• Nat Struct Mol Biol. 2008; 15: 777-9

• Khaperskyy DA, Ammerman ML, Majovski RC, Ponticelli AS
• Functions of Saccharomyces cerevisiae TFIIF during transcription startsite utilization.
• Mol Cell Biol. 2008; 28: 3757-66
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• Previous studies have shown that substitutions in the Tfg1 or Tfg2subunits of Saccharomyces cerevisiae transcription factor IIF (TFIIF) cancause upstream shifts in start site utilization, resulting in initiationpatterns that more closely resemble those of higher eukaryotes. In thisstudy, we report the results from multiple biochemical assays analyzingthe activities of wild-type yeast TFIIF and the TFIIF Tfg1 mutantcontaining the E346A substitution (Tfg1-E346A). We demonstrate that TFIIFstimulates formation of the first two phosphodiester bonds anddramatically stabilizes a short RNA-DNA hybrid in the RNA polymerase II(RNAPII) active center and, importantly, that the Tfg1-E346A substitutioncoordinately enhances early bond formation and the processivity of earlyelongation in vitro. These results are discussed within a proposed modelfor the role of yeast TFIIF in modulating conformational changes in theRNAPII active center during initiation and early elongation.

• Kvaratskhelia M, Grice SF
• Structural analysis of protein-RNA interactions with mass spectrometry.
• Methods Mol Biol. 2008; 488: 213-9
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• We present a high-resolution mass spectrometric footprinting approachenabling the identification of amino acids in the protein of interestinteracting with cognate RNA. This approach is particularly attractive forstudying large nucleoprotein complexes that are less amenable tocrystallographic or nuclear magnetic resonance analysis. Importantly, ourmethodology allows examination of protein-RNA interactions underbiologically relevant conditions using limited amounts of protein andnucleic acid samples.

• Ghosh A, Shuman S, Lima CD
• The structure of Fcp1, an essential RNA polymerase II CTD phosphatase.
• Mol Cell. 2008; 32: 478-90
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• Kinases and phosphatases regulate mRNA synthesis and processing byphosphorylating and dephosphorylating the C-terminal domain (CTD) of thelargest subunit of RNA polymerase II. Fcp1 is an essential CTD phosphatasethat preferentially hydrolyzes Ser2-PO(4) of the tandem YSPTSPS CTD heptadarray. Fcp1 crystal structures were captured at two stages of the reactionpathway: a Mg-BeF(3) complex that mimics the aspartylphosphateintermediate and a Mg-AlF(4)(-) complex that mimics the transition stateof the hydrolysis step. Fcp1 is a Y-shaped protein composed of anacylphosphatase domain located at the base of a deep canyon formed byflanking modules that are missing from the small CTD phosphatase (SCP)clade: an Fcp1-specific helical domain and a C-terminal BRCA1 C-terminal(BRCT) domain. The structure and mutational analysis reveals that Fcp1 andScp1 (a Ser5-selective phosphatase) adopt different CTD-binding modes; wesurmise the CTD threads through the Fcp1 canyon to access the active site.

• Kornberg RD
• The molecular basis of eukaryotic transcription.
• Proc Natl Acad Sci U S A. 2007; 104: 12955-61

• Almeida MS, Johnson MA, Herrmann T, Geralt M, Wuthrich K
• Novel beta-barrel fold in the nuclear magnetic resonance structure of thereplicase nonstructural protein 1 from the severe acute respiratorysyndrome coronavirus.
• J Virol. 2007; 81: 3151-61
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• The nonstructural protein 1 (nsp1) of the severe acute respiratorysyndrome coronavirus has 179 residues and is the N-terminal cleavageproduct of the viral replicase polyprotein that mediates RNA replicationand processing. The specific function of nsp1 is not known. Here we reportthe nuclear magnetic resonance structure of the nsp1 segment from residue13 to 128, which represents a novel alpha/beta-fold formed by a mixedparallel/antiparallel six-stranded beta-barrel, an alpha-helix coveringone opening of the barrel, and a 3(10)-helix alongside the barrel. Wefurther characterized the full-length 179-residue protein and show thatthe polypeptide segments of residues 1 to 12 and 129 to 179 are flexiblydisordered. The structure is analyzed in a search for possiblecorrelations with the recently reported activity of nsp1 in thedegradation of mRNA.

• Vojnic E, Simon B, Strahl BD, Sattler M, Cramer P
• Structure and carboxyl-terminal domain (CTD) binding of the Set2 SRIdomain that couples histone H3 Lys36 methylation to transcription.
• J Biol Chem. 2006; 281: 13-5
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• During mRNA elongation, the SRI domain of the histone H3 methyltransferaseSet2 binds to the phosphorylated carboxyl-terminal domain (CTD) of RNApolymerase II. The solution structure of the yeast Set2 SRI domain revealsa novel CTD-binding fold consisting of a left-handed three-helix bundle.NMR titration shows that the SRI domain binds an Ser2/Ser5-phosphorylatedCTD peptide comprising two heptapeptide repeats and three flankingNH2-terminal residues, whereas a single CTD repeat is insufficient forbinding. Residues that show strong chemical shift perturbations upon CTDbinding cluster in two regions. Both CTD tyrosine side chains contact theSRI domain. One of the tyrosines binds in the region with the strongestchemical shift perturbations, formed by the two NH2-terminal helices.Unexpectedly, the SRI domain fold resembles the structure of an RNApolymerase-interacting domain in bacterial sigma factors (domain sigma2 insigma70).

• Goodwin TJ, Butler MI, Poulter RT
• Multiple, non-allelic, intein-coding sequences in eukaryotic RNApolymerase genes.
• BMC Biol. 2006; 4: 38-38
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• BACKGROUND: Inteins are self-splicing protein elements. They aretranslated as inserts within host proteins that excise themselves andligate the flanking portions of the host protein (exteins) with a peptidebond. They are encoded as in-frame insertions within the genes for thehost proteins. Inteins are found in all three domains of life and inviruses, but have a very sporadic distribution. Only a small number ofintein coding sequences have been identified in eukaryotic nuclear genes,and all of these are from ascomycete or basidiomycete fungi. RESULTS: Weidentified seven intein coding sequences within nuclear genes coding forthe second largest subunits of RNA polymerase. These sequences were foundin diverse eukaryotes: one is in the second largest subunit of RNApolymerase I (RPA2) from the ascomycete fungus Phaeosphaeria nodorum, oneis in the RNA polymerase III (RPC2) of the slime mould Dictyosteliumdiscoideum and four intein coding sequences are in RNA polymerase II genes(RPB2), one each from the green alga Chlamydomonas reinhardtii, thezygomycete fungus Spiromyces aspiralis and the chytrid fungiBatrachochytrium dendrobatidis and Coelomomyces stegomyiae. The remainingintein coding sequence is in a viral relic embedded within the genome ofthe oomycete Phytophthora ramorum. The Chlamydomonas and Dictyosteliuminteins are the first nuclear-encoded inteins found outside of the fungi.These new inteins represent a unique dataset: they are found in homologousproteins that form a paralogous group. Although these paralogues divergedearly in eukaryotic evolution, their sequences can be aligned over most oftheir length. The inteins are inserted at multiple distinct sites, each ofwhich corresponds to a highly conserved region of RNA polymerase. Thisdataset supports earlier work suggesting that inteins preferentially occurin highly conserved regions of their host proteins. CONCLUSION: Theidentification of these new inteins increases the known host range ofintein sequences in eukaryotes, and provides fresh insights into theirorigins and evolution. We conclude that inteins are ancient eukaryoteelements once found widely among microbial eukaryotes. They persist asrarities in the genomes of a sporadic array of microorganisms, occupyinghighly conserved sites in diverse proteins.

• Hoshino M et al.
• Transcriptional repression induces a slowly progressive atypical neuronaldeath associated with changes of YAP isoforms and p73.
• J Cell Biol. 2006; 172: 589-604
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• Transcriptional disturbance is implicated in the pathology ofpolyglutamine diseases, including Huntington's disease (HD). However, itis unknown whether transcriptional repression leads to neuronal death orwhat forms that death might take. We found transcriptionalrepression-induced atypical death (TRIAD) of neurons to be distinct fromapoptosis, necrosis, or autophagy. The progression of TRIAD was extremelyslow in comparison with other types of cell death. Gene expressionprofiling revealed the reduction of full-length yes-associated protein(YAP), a p73 cofactor to promote apoptosis, as specific to TRIAD.Furthermore, novel neuron-specific YAP isoforms (YAPDeltaCs) weresustained during TRIAD to suppress neuronal death in a dominant-negativefashion. YAPDeltaCs and activated p73 were colocalized in the striatalneurons of HD patients and mutant huntingtin (htt) transgenic mice.YAPDeltaCs also markedly attenuated Htt-induced neuronal death in primaryneuron and Drosophila melanogaster models. Collectively, transcriptionalrepression induces a novel prototype of neuronal death associated with thechanges of YAP isoforms and p73, which might be relevant to the HDpathology.

• Wang D, Bushnell DA, Westover KD, Kaplan CD, Kornberg RD
• Structural basis of transcription: role of the trigger loop in substratespecificity and catalysis.
• Cell. 2006; 127: 941-54
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• New structures of RNA polymerase II (pol II) transcribing complexes reveala likely key to transcription. The trigger loop swings beneath a correctnucleoside triphosphate (NTP) in the nucleotide addition site, closing offthe active center and forming an extensive network of interactions withthe NTP base, sugar, phosphates, and additional pol II residues. Ahistidine side chain in the trigger loop, precisely positioned by theseinteractions, may literally "trigger" phosphodiester bond formation.Recognition and catalysis are thus coupled, ensuring the fidelity oftranscription.

• Zhang C, Zobeck KL, Burton ZF
• Human RNA polymerase II elongation in slow motion: role of the TFIIF RAP74alpha1 helix in nucleoside triphosphate-driven translocation.
• Mol Cell Biol. 2005; 25: 3583-95
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• The role of the RAP74 alpha1 helix of transcription factor IIF (TFIIF) instimulating elongation by human RNA polymerase II (RNAP II) was examinedusing millisecond-phase transient-state kinetics. RAP74 deletion mutantsRAP74(1-227), which includes an intact alpha1 helix, and RAP74(1-158), inwhich the alpha1 helix is deleted, were compared. Analysis of TFIIFRAP74-RAP30 complexes carrying the RAP74(1-158) deletion reveals the roleof the alpha1 helix because this mutant has indistinguishable activitycompared to TFIIF 74(W164A), which carries a critical point mutation inalpha1. We report adequate two-bond kinetic simulations for the reactionin the presence of TFIIF 74(1-227) + TFIIS and TFIIF 74(1-158) + TFIIS.TFIIF 74(1-158) is defective because it fails to promote forwardtranslocation. Deletion of the RAP74 alpha1 helix results in increasedoccupancy of the backtracking, cleavage, and restart pathways at a stallposition, indicating reverse translocation of the elongation complex.During elongation, TFIIF 74(1-158) fails to support detectable nucleosidetriphosphate (NTP)-driven translocation from a stall position and isnotably defective in supporting bond completion (NTP-driven translocationcoupled to pyrophosphate release) during the processive transition betweenbonds.

• Ghosh M, Elsby LM, Mal TK, Gooding JM, Roberts SG, Ikura M
• Probing Zn2+-binding effects on the zinc-ribbon domain of human generaltranscription factor TFIIB.
• Biochem J. 2004; 378: 317-24
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• The general transcription factor, TFIIB, plays an important role in theassembly of the pre-initiation complex. The N-terminal domain (NTD) ofTFIIB contains a zinc-ribbon motif, which is responsible for therecruitment of RNA polymerase II and TFIIF to the core promoter region.Although zinc-ribbon motif structures of eukaryotic and archaeal TFIIBshave been reported previously, the structural role of Zn2 binding to TFIIBremains to be determined. In the present paper, we report NMR andbiochemical studies of human TFIIB NTD, which characterize the structureand dynamics of the TFIIB Zn2-binding domain in both Zn2-bound and -freestates. The NMR data show that, whereas the backbone fold of NTD ispre-formed in the apo state, Zn2 binding reduces backbone mobility in theb-turn (Arg28-Gly30), induces enhanced structural rigidity of thecharged-cluster domain in the central linker region of TFIIB and appends apositive surface charge within the Zn2-binding site. V8protease-sensitivity assays of full-length TFIIB support the Zn2-dependentstructural changes. These structural effects of Zn2 binding on TFIIB mayhave a critical role in interactions with its binding partners, such asthe Rpb1 subunit of RNA polymerase II.

• Artsimovitch I et al.
• Structural basis for transcription regulation by alarmone ppGpp.
• Cell. 2004; 117: 299-310
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• Guanosine-tetraphosphate (ppGpp) is a major regulator of stringentcontrol, an adaptive response of bacteria to amino acid starvation. The2.7 A resolution structure of the Thermus thermophilus RNA polymerase(RNAP) holoenzyme in complex with ppGpp reveals that ppGpp binds to thesame site near the active center in both independent RNAP molecules in thecrystal but in strikingly distinct orientations. Binding is symmetricalwith respect to the two diphosphates of ppGpp and is relaxed with respectto the orientation of the nucleotide base. Different modes of ppGppbinding are coupled with asymmetry of the active site configurations. Theresults suggest that base pairing of ppGpp with cytosines in thenontemplate DNA strand might be an essential component of transcriptioncontrol by ppGpp. We present experimental evidence highlighting theimportance of base-specific contacts between ppGpp and specific cytosineresidues during both transcription initiation and elongation.

• Bushnell DA, Westover KD, Davis RE, Kornberg RD
• Structural basis of transcription: an RNA polymerase II-TFIIB cocrystal at4.5 Angstroms.
• Science. 2004; 303: 983-8
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• The structure of the general transcription factor IIB (TFIIB) in a complexwith RNA polymerase II reveals three features crucial for transcriptioninitiation: an N-terminal zinc ribbon domain of TFIIB that contacts the"dock" domain of the polymerase, near the path of RNA exit from atranscribing enzyme; a "finger" domain of TFIIB that is inserted into thepolymerase active center; and a C-terminal domain, whose interaction withboth the polymerase and with a TATA box-binding protein (TBP)-promoter DNAcomplex orients the DNA for unwinding and transcription. TFIIB stabilizesan early initiation complex, containing an incomplete RNA-DNA hybridregion. It may interact with the template strand, which sets the locationof the transcription start site, and may interfere with RNA exit, whichleads to abortive initiation or promoter escape. The trajectory ofpromoter DNA determined by the C-terminal domain of TFIIB traverses sitesof interaction with TFIIE, TFIIF, and TFIIH, serving to define their rolesin the transcription initiation process.

• Campbell JW, Hodgson GI, Watson HC
• Low resolution structure of yeast phosphoglycerate mutase.
• Nat New Biol. 1972; 240: 137-9