NORMAL GENOMIC

• Kwapisz M, Beckouet F, Thuriaux P
• Early evolution of eukaryotic DNA-dependent RNA polymerases.
• Trends Genet. 2008; 24: 211-5
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• Eukaryotic DNA-dependent RNA polymerases (Pol I-III) share a conservedcore of 12 subunits, which is closely related to archaeal RNA polymerases.Rpb8, a subunit found in Pol I, II and III, was thought to be restrictedto eukaryotes. We show here that Rpb8 closely resembles an archaealprotein called G, found only in Crenarchaea, which identifies a lastmissing link between the core structure of archaeal and eukaryotic RNApolymerases.

• Knutson BA, Broyles SS
• Expansion of poxvirus RNA polymerase subunits sharing homology withcorresponding subunits of RNA polymerase II.
• Virus Genes. 2008; 36: 307-11
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• Poxvirus-encoded RNA polymerases were known previously to share extensivesequence homology in their two largest subunits with the correspondingsubunits of cellular RNA polymerases and a modest alignment between thesmallest poxvirus subunit and RBP10 of RNA polymerase II. The remainingsubunits had no apparent cellular homologs. In this study, the HHpredprogram that combines amino acid sequence alignments with secondarystructure predictions was used to search for homologs to the poxvirus RNApolymerase subunits. Significant matches of vaccinia RNA polymerase 22-,19-, and 18-kDa subunits to RNA polymerase II subunits RPB5, 6, and 7,respectively, were identified. These results strengthen the concept thatpoxviral RNA polymerases likely evolved from cellular RNA polymerases.

• Wigneshweraraj SR, Burrows PC, Severinov K, Buck M
• Stable DNA opening within open promoter complexes is mediated by the RNApolymerase beta'-jaw domain.
• J Biol Chem. 2005; 280: 36176-84
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• DNA opening for transcription-competent open promoter complex (OC)formation by the bacterial RNA polymerase (RNAP) relies upon a complexnetwork of interactions between the structurally conserved and flexiblemodules of the catalytic beta and beta'-subunits, RNAP-associatedsigma-subunit, and the DNA. Here, we show that one such module, thebeta'-jaw, functions to stabilize the OC. In OCs formed by the majorsigma70-RNAP, the stabilizing role of the beta'-jaw is not restricted toany particular melted DNA segment. In contrast, in OCs formed by the majorvariant sigma54-RNAP, the beta'-jaw and a conserved sigma54 regulatorydomain co-operate to stabilize the melted DNA segment immediately upstreamof the transcription start site. Clearly, regulated communication betweenthe mobile modules of the RNAP and the functional domain(s) of the sigmasubunit is required for stable DNA opening.

• Artsimovitch I, Svetlov V, Anthony L, Burgess RR, Landick R
• RNA polymerases from Bacillus subtilis and Escherichia coli differ inrecognition of regulatory signals in vitro.
• J Bacteriol. 2000; 182: 6027-35
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• Adaptation of bacterial cells to diverse habitats relies on the ability ofRNA polymerase to respond to various regulatory signals. Some of thesesignals are conserved throughout evolution, whereas others are speciesspecific. In this study we present a comprehensive comparative analysis ofRNA polymerases from two distantly related bacterial species, Escherichiacoli and Bacillus subtilis, using a panel of in vitro transcriptionassays. We found substantial species-specific differences in the abilityof these enzymes to escape from the promoter and to recognize certaintypes of elongation signals. Both enzymes responded similarly to otherpause and termination signals and to the general E. coli elongationfactors NusA and GreA. We also demonstrate that, although promoterrecognition depends largely on the sigma subunit, promoter discriminationexhibited in species-specific fashion by both RNA polymerases resides inthe core enzyme. We hypothesize that differences in signal recognition aredue to the changes in contacts made between the beta and beta' subunitsand the downstream DNA duplex.

• Szilvay AM, Stern B, Blichenberg A, Helland DE
• Structural and functional similarities between HIV-1 reverse transcriptaseand the Escherichia coli RNA polymerase beta' subunit.
• FEBS Lett. 2000; 484: 43-7
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• Four monoclonal antibodies (MAbs) recognizing HIV-1 reverse transcriptase(RT) were shown here to cross-react with the beta' subunit of Escherichiacoli RNA polymerase (RNAP). The anti-RT MAbs bind to a peptide comprisingresidues 294-305 of the RT amino acid sequence. Computer analyses revealedsequence similarity between this peptide and two regions of the RNAP beta'subunit. MAb-binding studies using RT mutants suggested that the epitopeis located to amino acids 652-663 of the beta' sequence. One of the MAbswhich inhibited the polymerase activity of RT also mediated a dosedependent inhibition of the RNAP activity.

• Nakasone K, Takaki Y, Takami H, Inoue A, Horikoshi K
• Cloning and expression of the gene encoding RNA polymerase alpha subunitfrom alkaliphilic Bacillus sp. strain C-125.
• FEMS Microbiol Lett. 1998; 168: 269-76
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• The rpoA gene, encoding the alpha subunit of RNA polymerase, was isolatedfrom alkaliphilic Bacillus sp. strain C-125 by the PCR method. A 3-kbHindIII fragment containing the complete rpoA gene was cloned andsequenced. The alpha subunit gene was found to encode a protein consistingof 314 amino acid residues with a molecular mass of 34,805 Da. Comparedwith the amino acid sequences of other known eubacterial RNA polymerasealpha subunits, the gene has 84% identity to that of B. subtilis, whileshowing 48% and 47% identity to that of Streptomyces coelicolor andEscherichia coli, respectively. Six conserved regions, which are observedin the case of other eubacteria, were found in the RNA polymerase alphasubunit of this strain. Five of them are located in the N-terminal domaininvolved in assembly of the core enzyme, while one is located in theC-terminal domain, which interacts with several transcriptional factorsand a specific DNA element. By means of recombinant plasmids, ahexahistidine-tagged derivative of the RNA polymerase alpha subunit ofstrain C-125 and two deletion derivatives (C- and N-terminal domains) ofthis protein were overexpressed in E. coli cells and purified to nearhomogeneity.

• Engel JN, Pollack J, Malik F, Ganem D
• Cloning and characterization of RNA polymerase core subunits of Chlamydiatrachomatis by using the polymerase chain reaction.
• J Bacteriol. 1990; 172: 5732-41
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• Taking advantage of sequence conservation of portions of the alpha, beta,and beta' subunits of RNA polymerase of bacteria and plant chloroplasts,we have designed degenerate oligonucleotides corresponding to thesedomains and used these synthetic DNA sequences as primers in a polymerasechain reaction to amplify DNA sequences from the chlamydial genome. Thepolymerase chain reaction products were used as a probe to recover thegenomic fragments encoding the beta subunit and the 5' portion of thebeta' subunit from a library of cloned murine Chlamydia trachomatis DNA.Similar attempts to recover the alpha subunit were unsuccessful. Sequenceanalysis demonstrated that the beta subunit of RNA polymerase was locatedbetween genes encoding the L7/L12 ribosomal protein and the beta' subunitof RNA polymerase; this organization is reminiscent of the rpoBC operon ofEscherichia coli. The C. trachomatis beta subunit overproduced in E. coliwas used as an antigen in rabbits to make a polyclonal antibody to thissubunit. Although this polyclonal antibody specifically immunoprecipitatedthe beta subunit from Chlamydia-infected cells, it did notimmunoprecipitate core or holoenzyme. Immunoblots with this antibodydemonstrated that the beta subunit appeared early in infection.

• Grachev MA, Lukhtanov EA, Mustaev AA, Abdukaiumov MN, Rabinov IV
• [Localization of a histidine residue in the binding site for theinitiating substrate of E. coli RNA-polymerase].
• Bioorg Khim. 1987; 13: 992-5
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• E. coli RNA polymerase was selectively labelled in the presence ofpromoters at a histidine residue of the beta-subunit by treatment with GDPbeta-imidazolide and then with [alpha-32P]UTP (or [alpha-33P]UTP). Partialcyanogen bromide cleavage of the labelled polypeptide afforded a series of"single-hit" labelled peptides, the electrophoretic pattern of whichsuggested that the labelling site was His1237. This conclusion wasconfirmed by a similar pattern obtained with products of the cyanogenbromide cleavage of a radioactive peptide obtained by the limitedtrypsinolysis (C-terminal peptide consisting of 423 amino acid residues).Interpretation of our earlier results in favour of His1116 as thelabelling point (Dokl. Acad. nauk SSSR, 1985, v. 281, p. 723) wasincorrect due to the electrophoretic "compression" of three labelledpeptide bands.

• Kalyaeva ES, Sever IS, Nikiforov VG, Danilevskaya ON
• A mutation suppressing the overproduction of RNA polymerase beta beta'subunits in the RpoC1 strain of Escherichia coli.
• Mol Gen Genet. 1980; 178: 669-74

• Ovchinnikov YA, Lipkin VM, Modyanov NN, Chertov OY, Smirnov YV
• Primary structure of alpha-subunit of DNA-dependent RNA polymerase fromEscherichia coli.
• FEBS Lett. 1977; 76: 108-11

• Lowe PA, Malcolm AD
• The renaturation of subunits and subunit fragments of escherichia coliribonucleic acid polymerase.
• Biochem Soc Trans. 1975; 3: 652-3

• Kirschbaum JB, Scaife J
• Evidence for a lambda transducing phage carrying the genes for the betaand beta' subunits of Escherichia coli RNA polymerase.
• Mol Gen Genet. 1974; 132: 193-201