RNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism by which a helicase-like factor HelD recycles RNAP. We report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNAP channels that are responsible for nucleic acids binding and substrate delivery to the active site, thereby locking RNAP in an inactive state. We show that HelD prevents non-specific interactions between RNAP and DNA and dissociates stalled transcription elongation complexes. The liberated RNAP can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that releases RNAP from nonfunctional complexes with nucleic acids.

CEITEC Masaryk University Brno Czech Republic

Department of Biochemistry and Molecular Biology The Center for RNA Molecular Biology Pennsylvania State University University Park PA 16802 USA

EMBL Grenoble 71 Avenue des Martyrs Grenoble France

Faculty of Mathematics and Physics Institute of Physics Charles University Prague Czech Republic

Institute of Biotechnology of the Czech Academy of Sciences Centre BIOCEV Průmyslová 595 252 50 Vestec Czech Republic

Institute of Microbiology of The Czech Academy of Sciences Prague Czech Republic

Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Kouba, T. et al. The core and holoenzyme forms of RNA polymerase from Mycobacterium smegmatis. J. Bacteriol. 201, 10.1128/JB.00583-18 (2019). PubMed PMC

Paget MS. Bacterial sigma factors and anti-sigma factors: structure, function and distribution. Biomolecules. 2015;5:1245–1265. doi: 10.3390/biom5031245. PubMed DOI PMC

Lee J, Borukhov S. Bacterial RNA polymerase-DNA interaction-the driving force of gene expression and the target for drug action. Front. Mol. Biosci. 2016;3:73. PubMed PMC

Barvik I, Rejman D, Panova N, Sanderova H, Krasny L. Non-canonical transcription initiation: the expanding universe of transcription initiating substrates. FEMS Microbiol. Rev. 2017;41:131–138. PubMed

Vassylyev DG, Vassylyeva MN, Perederina A, Tahirov TH, Artsimovitch I. Structural basis for transcription elongation by bacterial RNA polymerase. Nature. 2007;448:157–162. doi: 10.1038/nature05932. PubMed DOI

Lopez de Saro FJ, Yoshikawa N, Helmann JD. Expression, abundance, and RNA polymerase binding properties of the delta factor of Bacillus subtilis. J. Biol. Chem. 1999;274:15953–15958. doi: 10.1074/jbc.274.22.15953. PubMed DOI

Keller AN, et al. epsilon, a new subunit of RNA polymerase found in gram-positive bacteria. J. Bacteriol. 2014;196:3622–3632. doi: 10.1128/JB.02020-14. PubMed DOI PMC

Jensen D, Manzano AR, Rammohan J, Stallings CL, Galburt EA. CarD and RbpA modify the kinetics of initial transcription and slow promoter escape of the Mycobacterium tuberculosis RNA polymerase. Nucleic Acids Res. 2019;47:6685–6698. doi: 10.1093/nar/gkz449. PubMed DOI PMC

Delumeau O, et al. The dynamic protein partnership of RNA polymerase in Bacillus subtilis. Proteomics. 2011;11:2992–3001. doi: 10.1002/pmic.201000790. PubMed DOI

Fairman-Williams ME, Guenther UP, Jankowsky E. SF1 and SF2 helicases: family matters. Curr. Opin. Struct. Biol. 2010;20:313–324. doi: 10.1016/j.sbi.2010.03.011. PubMed DOI PMC

Wiedermannova J, et al. Characterization of HelD, an interacting partner of RNA polymerase from Bacillus subtilis. Nucleic Acids Res. 2014;42:5151–5163. doi: 10.1093/nar/gku113. PubMed DOI PMC

Koval T, et al. Domain structure of HelD, an interaction partner of Bacillus subtilis RNA polymerase. FEBS Lett. 2019;593:996–1005. doi: 10.1002/1873-3468.13385. PubMed DOI

Meeske AJ, et al. High-throughput genetic screens identify a large and diverse collection of new sporulation genes in Bacillus subtilis. PLoS Biol. 2016;14:e1002341. doi: 10.1371/journal.pbio.1002341. PubMed DOI PMC

Krissinel E, Henrick K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 2007;372:774–797. doi: 10.1016/j.jmb.2007.05.022. PubMed DOI

Ross W, et al. ppGpp binding to a site at the RNAP-DksA interface accounts for its dramatic effects on transcription initiation during the stringent response. Mol. Cell. 2016;62:811–823. doi: 10.1016/j.molcel.2016.04.029. PubMed DOI PMC

Molodtsov V, et al. Allosteric effector ppGpp potentiates the inhibition of transcript initiation by DksA. Mol. Cell. 2018;69:828–839. doi: 10.1016/j.molcel.2018.01.035. PubMed DOI PMC

Abdelkareem M, et al. Structural basis of transcription: RNA polymerase backtracking and its reactivation. Mol. Cell. 2019;75:298–309. doi: 10.1016/j.molcel.2019.04.029. PubMed DOI PMC

Laptenko O, Lee J, Lomakin I, Borukhov S. Transcript cleavage factors GreA and GreB act as transient catalytic components of RNA polymerase. EMBO J. 2003;22:6322–6334. doi: 10.1093/emboj/cdg610. PubMed DOI PMC

Perederina A, et al. Regulation through the secondary channel–structural framework for ppGpp-DksA synergism during transcription. Cell. 2004;118:297–309. doi: 10.1016/j.cell.2004.06.030. PubMed DOI

Sosunova E, et al. Donation of catalytic residues to RNA polymerase active center by transcription factor Gre. Proc. Natl Acad. Sci. USA. 2003;100:15469–15474. doi: 10.1073/pnas.2536698100. PubMed DOI PMC

Raney KD, Byrd AK, Aarattuthodiyil S. Structure and mechanisms of SF1 DNA helicases. Adv. Exp. Med. Biol. 2013;973:E1. 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. doi: 10.1016/j.cell.2006.10.049. PubMed DOI PMC

Liu B, Zuo Y, Steitz TA. Structural basis for transcription reactivation by RapA. Proc. Natl Acad. Sci. USA. 2015;112:2006–2010. doi: 10.1073/pnas.1417152112. PubMed DOI PMC

Tafur L, et al. Molecular structures of transcribing RNA polymerase I. Mol. Cell. 2016;64:1135–1143. doi: 10.1016/j.molcel.2016.11.013. PubMed DOI PMC

Chen J, et al. Stepwise promoter melting by bacterial RNA polymerase. Mol. Cell. 2020;78:275–288. doi: 10.1016/j.molcel.2020.02.017. PubMed DOI PMC

Lin W, et al. Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3) Mol. Cell. 2018;70:60–71. doi: 10.1016/j.molcel.2018.02.026. PubMed DOI PMC

Boyaci, H. et al. Fidaxomicin jams Mycobacterium tuberculosis RNA polymerase motions needed for initiation via RbpA contacts. Elife7, 10.7554/eLife.34823 (2018). PubMed PMC

Kohler R, Mooney RA, Mills DJ, Landick R, Cramer P. Architecture of a transcribing-translating expressome. Science. 2017;356:194–197. doi: 10.1126/science.aal3059. PubMed DOI PMC

Pani B, Nudler E. Mechanistic insights into transcription coupled DNA repair. DNA Repair. 2017;56:42–50. doi: 10.1016/j.dnarep.2017.06.006. PubMed DOI PMC

Lang KS, Merrikh H. The clash of macromolecular titans: replication-transcription conflicts in bacteria. Annu. Rev. Microbiol. 2018;72:71–88. doi: 10.1146/annurev-micro-090817-062514. PubMed DOI PMC

Newing, T. et al. Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD. Nature Commun.10.1038/s41467-020-20157-5 (2020). PubMed PMC

Pei, H.-H. et al. The δ subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling. Nature Commun.10.1038/s41467-020-20159-3 (2020). PubMed PMC

Harden TT, et al. Alternative transcription cycle for bacterial RNA polymerase. Nat. Commun. 2020;11:448. doi: 10.1038/s41467-019-14208-9. PubMed DOI PMC

Tran QH, Unden G. Changes in the proton potential and the cellular energetics of Escherichia coli during growth by aerobic and anaerobic respiration or by fermentation. Eur. J. Biochem. 1998;251:538–543. doi: 10.1046/j.1432-1327.1998.2510538.x. PubMed DOI

Chen J, et al. 6S RNA mimics B-form DNA to regulate Escherichia coli RNA polymerase. Mol. Cell. 2017;68:388–397. doi: 10.1016/j.molcel.2017.09.006. PubMed DOI PMC

Hnilicova J, et al. Ms1, a novel sRNA interacting with the RNA polymerase core in mycobacteria. Nucleic Acids Res. 2014;42:11763–11776. doi: 10.1093/nar/gku793. PubMed DOI PMC

Choudhary E, Thakur P, Pareek M, Agarwal N. Gene silencing by CRISPR interference in mycobacteria. Nat. Commun. 2015;6:6267. doi: 10.1038/ncomms7267. PubMed DOI

Zhang K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 2016;193:1–12. doi: 10.1016/j.jsb.2015.11.003. PubMed DOI PMC

Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife7, 10.7554/eLife.42166 (2018). PubMed PMC

Tegunov D, Cramer P. Real-time cryo-electron microscopy data preprocessing with Warp. Nat. Methods. 2019;16:1146–1152. doi: 10.1038/s41592-019-0580-y. PubMed DOI PMC

Wilkinson ME, Kumar A, Casanal A. Methods for merging data sets in electron cryo-microscopy. Acta Crystallogr. D Struct. Biol. 2019;75:782–791. doi: 10.1107/S2059798319010519. PubMed DOI PMC

Zivanov J, Nakane T, Scheres SHW. Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1. IUCrJ. 2020;7:253–267. doi: 10.1107/S2052252520000081. PubMed DOI PMC

Burnley T, Palmer CM, Winn M. Recent developments in the CCP-EM software suite. Acta Crystallogr. D Struct. Biol. 2017;73:469–477. doi: 10.1107/S2059798317007859. PubMed DOI PMC

Jakobi, A. J., Wilmanns, M. & Sachse, C. Model-based local density sharpening of cryo-EM maps. Elife6, 10.7554/eLife.27131 (2017). PubMed PMC

Tan YZ, et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods. 2017;14:793–796. doi: 10.1038/nmeth.4347. PubMed DOI PMC

Vagin A, Teplyakov A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 2010;66:22–25. doi: 10.1107/S0907444909042589. PubMed DOI

Brown A, et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D Biol. Crystallogr. 2015;71:136–153. doi: 10.1107/S1399004714021683. PubMed DOI PMC

Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 2004;60:2126–2132. doi: 10.1107/S0907444904019158. PubMed DOI

Cowtan K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006;62:1002–1011. doi: 10.1107/S0907444906022116. PubMed DOI

Terashi G, Kihara D. De novo main-chain modeling for EM maps using MAINMAST. Nat. Commun. 2018;9:1618. doi: 10.1038/s41467-018-04053-7. PubMed DOI PMC

Afonine PV, et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D Struct. Biol. 2018;74:531–544. doi: 10.1107/S2059798318006551. PubMed DOI PMC

Jurrus E, et al. Improvements to the APBS biomolecular solvation software suite. Protein Sci. 2018;27:112–128. doi: 10.1002/pro.3280. PubMed DOI PMC

Pettersen EF, et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 2004;25:1605–1612. doi: 10.1002/jcc.20084. PubMed DOI

McNicholas S, Potterton E, Wilson KS, Noble ME. Presenting your structures: the CCP4mg molecular-graphics software. Acta Crystallogr. D Biol. Crystallogr. 2011;67:386–394. doi: 10.1107/S0907444911007281. PubMed DOI PMC

Schlee S, et al. Prediction of quaternary structure by analysis of hot spot residues in protein-protein interfaces: the case of anthranilate phosphoribosyltransferases. Proteins. 2019;87:815–825. doi: 10.1002/prot.25744. PubMed DOI

Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997;276:307–326. doi: 10.1016/S0076-6879(97)76066-X. PubMed DOI

He Z, Honeycutt CW. A modified molybdenum blue method for orthophosphate determination suitable for investigating enzymatic hydrolysis of organic phosphates. Commun. Soil Sci. Plant Anal. 2005;36:1373–1383. doi: 10.1081/CSS-200056954. DOI

Komissarova N, Kireeva ML, Becker J, Sidorenkov I, Kashlev M. Engineering of elongation es of bacterial and yeast RNA polymerases. Methods Enzymol. 2003;371:233–251. doi: 10.1016/S0076-6879(03)71017-9. PubMed DOI

Sikova M, et al. The torpedo effect in Bacillus subtilis: RNase J1 resolves stalled transcription complexes. EMBO J. 2020;39:e102500. doi: 10.15252/embj.2019102500. PubMed DOI PMC

Deng Z, et al. Yin Yang 1 regulates the transcriptional activity of androgen receptor. Oncogene. 2009;28:3746–3757. doi: 10.1038/onc.2009.231. PubMed DOI PMC

MoaB2, a newly identified transcription factor, binds to σA in Mycobacterium smegmatis

Mycobacterial HelD connects RNA polymerase recycling with transcription initiation

RIP-seq reveals RNAs that interact with RNA polymerase and primary sigma factors in bacteria

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