Bacterial RNA polymerase (RNAP) is an essential multisubunit protein complex required for gene expression. Here, we characterize YvgS (HelD) from Bacillus subtilis, a novel binding partner of RNAP. We show that HelD interacts with RNAP-core between the secondary channel of RNAP and the alpha subunits. Importantly, we demonstrate that HelD stimulates transcription in an ATP-dependent manner by enhancing transcriptional cycling and elongation. We demonstrate that the stimulatory effect of HelD can be amplified by a small subunit of RNAP, delta. In vivo, HelD is not essential but it is required for timely adaptations of the cell to changing environment. In summary, this study establishes HelD as a valid component of the bacterial transcription machinery.

Laboratory of Molecular Genetics of Bacteria Institute of Microbiology Academy of Sciences of the Czech Republic Prague 14220 Czech Republic Department of Genetics and Microbiology Faculty of Science Charles University Prague Prague 12843 Czech Republic Department of Structure Analysis of Biomacromolecules Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Prague 16206 Czech Republic Laboratory of Structure and Function of Biomolecules Institute of Biotechnology Academy of Sciences of the Czech Republic Prague 14220 Czech Republic School of Environmental and Life Sciences University of Newcastle Callaghan NSW 2308 Australia and Laboratory of Molecular Structure Characterization Institute of Microbiology Academy of Sciences of the Czech Republic Prague 14220 Czech Republic

Zobrazit více v PubMed

Osterberg S, del Peso-Santos T, Shingler V. Regulation of alternative sigma factor use. Annu. Rev. Microbiol. 2011;65:37–55. PubMed

Juang YL, Helmann JD. The delta subunit of Bacillus subtilis RNA polymerase. An allosteric effector of the initiation and core-recycling phases of transcription. J. Mol. Biol. 1994;239:1–14. PubMed

Lopez de Saro FJ, Woody AY, Helmann JD. Structural analysis of the Bacillus subtilis delta factor: a protein polyanion which displaces RNA from RNA polymerase. J. Mol. Biol. 1995;252:189–202. PubMed

Epshtein V, Dutta D, Wade J, Nudler E. An allosteric mechanism of Rho-dependent transcription termination. Nature. 2010;463:245–249. PubMed PMC

Toulme F, Mosrin-Huaman C, Sparkowski J, Das A, Leng M, Rahmouni AR. GreA and GreB proteins revive backtracked RNA polymerase in vivo by promoting transcript trimming. EMBO J. 2000;19:6853–6859. PubMed PMC

Yang X, Molimau S, Doherty GP, Johnston EB, Marles-Wright J, Rothnagel R, Hankamer B, Lewis RJ, Lewis PJ. The structure of bacterial RNA polymerase in complex with the essential transcription elongation factor NusA. EMBO Rep. 2009;10:997–1002. PubMed PMC

Delumeau O, Lecointe F, Muntel J, Guillot A, Guedon E, Monnet V, Hecker M, Becher D, Polard P, Noirot P. The dynamic protein partnership of RNA polymerase in Bacillus subtilis. Proteomics. 2011;11:2992–3001. PubMed

Yang W. Lessons learned from UvrD helicase: mechanism for directional movement. Annu. Rev. Biophys. 2010;39:367–385. PubMed PMC

Nicolas P, Mader U, Dervyn E, Rochat T, Leduc A, Pigeonneau N, Bidnenko E, Marchadier E, Hoebeke M, Aymerich S, et al. Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis. Science. 2012;335:1103–1106. PubMed

Carrasco B, Fernandez S, Petit MA, Alonso JC. Genetic recombination in Bacillus subtilis 168: effect of DeltahelD on DNA repair and homologous recombination. J. Bacteriol. 2001;183:5772–5777. PubMed PMC

Hanahan D. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 1983;166:557–580. PubMed

Dubnau D, Davidoff-Abelson R. Fate of transforming DNA following uptake by competent Bacillus. subtilis. I. Formation and properties of the donor-recipient complex. J. Mol. Biol. 1971;56:209–221. PubMed

Qi Y, Hulett FM. PhoP-P and RNA polymerase sigmaA holoenzyme are sufficient for transcription of Pho regulon promoters in Bacillus subtilis: PhoP-P activator sites within the coding region stimulate transcription in vitro. Mol. Microbiol. 1998;28:1187–1197. PubMed

Kobayashi K, Ehrlich SD, Albertini A, Amati G, Andersen KK, Arnaud M, Asai K, Ashikaga S, Aymerich S, Bessieres P, et al. Essential Bacillus subtilis genes. Proc. Natl Acad. Sci. USA. 2003;100:4678–4683. PubMed PMC

Rabatinova A, Sanderova H, Matejckova JJ, Korelusova J, Sojka L, Barvik I, Papouskova V, Sklenar V, Zidek L, Krasny L. The delta subunit of RNA polymerase is required for rapid changes in gene expression and competitive fitness of the cell. J. Bacteriol. 2013;195:2603–2611. PubMed PMC

Chang BY, Doi RH. Overproduction, purification, and characterization of Bacillus subtilis RNA polymerase sigma A factor. J. Bacteriol. 1990;172:3257–3263. PubMed PMC

Ross W, Thompson JF, Newlands JT, Gourse RL. E. coli Fis protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J. 1990;9:3733–3742. PubMed PMC

Krasny L, Gourse RL. An alternative strategy for bacterial ribosome synthesis: Bacillus subtilis rRNA transcription regulation. EMBO J. 2004;23:4473–4483. PubMed PMC

Johnston EB, Lewis PJ, Griffith R. The interaction of Bacillus subtilis sigmaA with RNA polymerase. Protein Sci. 2009;18:2287–2297. PubMed PMC

Anborgh PH, Parmeggiani A, Jonak J. Site-directed mutagenesis of elongation factor Tu. The functional and structural role of residue Cys81. Eur. J. Biochem. 1992;208:251–257. PubMed

Gaal T, Ross W, Estrem ST, Nguyen LH, Burgess RR, Gourse RL. Promoter recognition and discrimination by EsigmaS RNA polymerase. Mol. Microbiol. 2001;42:939–954. PubMed

Barker MM, Gaal T, Gourse RL. Mechanism of regulation of transcription initiation by ppGpp. II. Models for positive control based on properties of RNAP mutants and competition for RNAP. J. Mol. Biol. 2001;305:689–702. PubMed

Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. BLAST+: architecture and applications. BMC Bioinformatics. 2008;10:421. PubMed PMC

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J. Mol. Biol. 1990;215:403–410. PubMed

Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23:2947–2948. PubMed

Anthony LC, Artsimovitch I, Svetlov V, Landick R, Burgess RR. Rapid purification of His(6)-tagged Bacillus subtilis core RNA polymerase. Protein Expr. Purif. 2000;19:350–354. PubMed

Lee JY, Yang W. UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke. Cell. 2006;127:1349–1360. PubMed PMC

Cao Z, Julin DA. Characterization in vitro and in vivo of the DNA helicase encoded by Deinococcus radiodurans locus DR1572. DNA Repair (Amst) 2009;8:612–619. PubMed

Vassylyev DG, Sekine S, Laptenko O, Lee J, Vassylyeva MN, Borukhov S, Yokoyama S. Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 A resolution. Nature. 2002;417:712–719. PubMed

Sukhodolets MV, Cabrera JE, Zhi H, Jin DJ. RapA, a bacterial homolog of SWI2/SNF2, stimulates RNA polymerase recycling in transcription. Genes Dev. 2001;15:3330–3341. PubMed PMC

Epshtein V, Toulme F, Rahmouni AR, Borukhov S, Nudler E. Transcription through the roadblocks: the role of RNA polymerase cooperation. EMBO J. 2003;22:4719–4727. PubMed PMC

Holmberg C, Rutberg B. Expression of the gene encoding glycerol-3-phosphate dehydrogenase (glpD) in Bacillus subtilis is controlled by antitermination. Mol. Microbiol. 1991;5:2891–2900. PubMed

Gowrishankar J, Leela JK, Anupama K. R-loops in bacterial transcription: their causes and consequences. Transcription. 2013;4:153–157. PubMed PMC

McKinley BA, Sukhodolets MV. Escherichia coli RNA polymerase-associated SWI/SNF protein RapA: evidence for RNA-directed binding and remodeling activity. Nucleic Acids Res. 2007;35:7044–7060. PubMed PMC

Vassylyev DG, Vassylyeva MN, Zhang J, Palangat M, Artsimovitch I, Landick R. Structural basis for substrate loading in bacterial RNA polymerase. Nature. 2007;448:163–168. PubMed

Yawn B, Zhang L, Mura C, Sukhodolets MV. RapA, the SWI/SNF subunit of Escherichia coli RNA polymerase, promotes the release of nascent RNA from transcription complexes. Biochemistry. 2009;48:7794–7806. PubMed PMC

Sukhodolets MV, Jin DJ. Interaction between RNA polymerase and RapA, a bacterial homolog of the SWI/SNF protein family. J. Biol. Chem. 2000;275:22090–22097. PubMed

Sukhodolets MV, Jin DJ. RapA, a novel RNA polymerase-associated protein, is a bacterial homolog of SWI2/SNF2. J. Biol. Chem. 1998;273:7018–7023. PubMed

Stec-Dziedzic E, Lyzen R, Skarfstad E, Shingler V, Szalewska-Palasz A. Characterization of the transcriptional stimulatory properties of the Pseudomonas putida RapA protein. Biochim. Biophys. Acta. 2013;1829:219–230. PubMed

Baranello L, Kouzine F, Levens D. DNA Topoisomerases: beyond the standard role. Transcription. 2013 4 doi: 10.4161/trns.26598. PubMed

Gwynn EJ, Smith AJ, Guy CP, Savery NJ, McGlynn P, Dillingham MS. The conserved C-terminus of the PcrA/UvrD helicase interacts directly with RNA polymerase. PLoS One. 2013;8:e78141. PubMed PMC

Mycobacterial HelD connects RNA polymerase recycling with transcription initiation

What the Hel: recent advances in understanding rifampicin resistance in bacteria

β-CASP proteins removing RNA polymerase from DNA: when a torpedo is needed to shoot a sitting duck

Quasi-essentiality of RNase Y in Bacillus subtilis is caused by its critical role in the control of mRNA homeostasis

Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

The torpedo effect in Bacillus subtilis: RNase J1 resolves stalled transcription complexes

Solution structure of domain 1.1 of the σA factor from Bacillus subtilis is preformed for binding to the RNA polymerase core

Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases

ε, a new subunit of RNA polymerase found in gram-positive bacteria