Mycobacterial HelD is a transcription factor that recycles stalled RNAP by dissociating it from nucleic acids and, if present, from the antibiotic rifampicin. The rescued RNAP, however, must disengage from HelD to participate in subsequent rounds of transcription. The mechanism of release is unknown. We show that HelD from Mycobacterium smegmatis forms a complex with RNAP associated with the primary sigma factor σA and transcription factor RbpA but not CarD. We solve several structures of RNAP-σA-RbpA-HelD without and with promoter DNA. These snapshots capture HelD during transcription initiation, describing mechanistic aspects of HelD release from RNAP and its protective effect against rifampicin. Biochemical evidence supports these findings, defines the role of ATP binding and hydrolysis by HelD in the process, and confirms the rifampicin-protective effect of HelD. Collectively, these results show that when HelD is present during transcription initiation, the process is protected from rifampicin until the last possible moment.

Department of Genetics and Microbiology Faculty of Science Charles University Viničná 5 128 44 Prague Czech Republic

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

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo náměstí 542 2 160 00 Prague Czech Republic

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Sutherland, C. & Murakami, K. S. An introduction to the structure and function of the catalytic core enzyme of PubMed PMC

Paget, M. S. Bacterial sigma factors and anti-sigma factors: structure, function and distribution. PubMed DOI PMC

Zhang, G. et al. Crystal structure of PubMed DOI

Browning, D. F. & Busby, S. J. Local and global regulation of transcription initiation in bacteria. PubMed DOI

Wassarman, K. M. 6S RNA, a Global Regulator of Transcription. PubMed PMC

Hnilicova, J. et al. Ms1, a novel sRNA interacting with the RNA polymerase core in mycobacteria. PubMed DOI PMC

Barvik, I., Rejman, D., Panova, N., Sanderova, H. & Krasny, L. Non-canonical transcription initiation: the expanding universe of transcription initiating substrates. PubMed

Koval, T. et al. Domain structure of HelD, an interaction partner of PubMed DOI

Kouba, T. et al. Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase. PubMed DOI PMC

Wiedermannova, J. et al. Characterization of HelD, an interacting partner of RNA polymerase from PubMed DOI PMC

Campbell, E. A. et al. Structural mechanism for rifampicin inhibition of bacterial rna polymerase. PubMed DOI

Larsen, J. S., Miller, M., Oakley, A. J., Dixon, N. E. & Lewis, P. J. Multiple classes and isoforms of the RNA polymerase recycling motor protein HelD. PubMed DOI PMC

Hurst-Hess, K. R., Saxena, A., Rudra, P., Yang, Y. & Ghosh, P. Mycobacterium abscessus HelR interacts with RNA polymerase to confer intrinsic rifamycin resistance. PubMed DOI PMC

Surette, M. D., Waglechner, N., Koteva, K. & Wright, G. D. HelR is a helicase-like protein that protects RNA polymerase from rifamycin antibiotics. PubMed DOI

Sudzinova, P. et al. What the Hel: recent advances in understanding rifampicin resistance in bacteria. PubMed PMC

Wilson, D. N., Hauryliuk, V., Atkinson, G. C. & O’Neill, A. J. Target protection as a key antibiotic resistance mechanism. PubMed DOI

Newing, T. P. et al. Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD. PubMed DOI PMC

Pei, H. H. et al. The delta subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling. PubMed DOI PMC

Wang, Z. et al. RbpA and sigma(B) association regulates polyphosphate levels to modulate mycobacterial isoniazid-tolerance. PubMed DOI

Hubin, E. A. et al. Structure and function of the mycobacterial transcription initiation complex with the essential regulator RbpA. PubMed PMC

Hu, Y. & Coates, A. R. Transcription of two sigma 70 homologue genes, sigA and sigB, in stationary-phase PubMed DOI PMC

Zhu, D. X. & Stallings, C. L. Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics. PubMed DOI PMC

Srivastava, D. B. et al. Structure and function of CarD, an essential mycobacterial transcription factor. PubMed DOI PMC

Pettersson, B. M. et al. Comparative sigma factor-mRNA levels in mycobacterium marinum under stress conditions and during host infection. PubMed DOI PMC

Hurst-Hess, K. et al. Mycobacterial SigA and SigB cotranscribe essential housekeeping genes during exponential growth. PubMed PMC

Singh, R. K. et al. Expression, purification, and in silico characterization of PubMed DOI PMC

Singha, B. et al. The unique N-terminal region of PubMed DOI PMC

Schwartz, E. C. et al. A full-length group 1 bacterial sigma factor adopts a compact structure incompatible with DNA binding. PubMed DOI PMC

Zachrdla, M. et al. Solution structure of domain 1.1 of the sigma(A) factor from Bacillus subtilis is preformed for binding to the RNA polymerase core. PubMed DOI PMC

Hubin, E. A., Lilic, M., Darst, S. A. & Campbell, E. A. Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures. PubMed DOI PMC

Morichaud, Z. et al. Structural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization. PubMed DOI PMC

Ruff, E. F., Record, M. T. Jr. & Artsimovitch, I. Initial events in bacterial transcription initiation. PubMed DOI PMC

Kouba, T. et al. The core and holoenzyme forms of RNA polymerase from PubMed PMC

Boyaci, H., Chen, J., Jansen, R., Darst, S. A. & Campbell, E. A. Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding. PubMed DOI PMC

Chen, J. et al. Stepwise promoter melting by bacterial RNA polymerase. PubMed DOI PMC

Feklistov, A. et al. RNA polymerase motions during promoter melting. PubMed DOI PMC

Chen, J. et al. PubMed PMC

Gulten, G. & Sacchettini, J. C. Structure of the Mtb CarD/RNAP beta-lobes complex reveals the molecular basis of interaction and presents a distinct DNA-binding domain for Mtb CarD. PubMed DOI PMC

Bae, B. et al. CarD uses a minor groove wedge mechanism to stabilize the RNA polymerase open promoter complex. PubMed PMC

Lee, J. Y. & Yang, W. UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke. PubMed DOI PMC

China, A., Tare, P. & Nagaraja, V. Comparison of promoter-specific events during transcription initiation in mycobacteria. PubMed DOI

Sojka, L. et al. Rapid changes in gene expression: DNA determinants of promoter regulation by the concentration of the transcription initiating NTP in PubMed DOI PMC

Krasny, L., Tiserova, H., Jonak, J., Rejman, D. & Sanderova, H. The identity of the transcription +1 position is crucial for changes in gene expression in response to amino acid starvation in PubMed DOI

Barker, M. M. & Gourse, R. L. Regulation of rRNA transcription correlates with nucleoside triphosphate sensing. PubMed DOI PMC

Zenkin, N. & Yuzenkova, Y. New insights into the functions of transcription factors that bind the RNA polymerase secondary channel. PubMed DOI PMC

Paul, B. J. et al. DksA: a critical component of the transcription initiation machinery that potentiates the regulation of rRNA promoters by ppGpp and the initiating NTP. PubMed DOI

Gopalkrishnan, S., Ross, W., Chen, A. Y. & Gourse, R. L. TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp. PubMed DOI PMC

Shin, Y. et al. Structural basis of ribosomal RNA transcription regulation. PubMed DOI PMC

He, D. et al. Pseudomonas aeruginosa SutA wedges RNAP lobe domain open to facilitate promoter DNA unwinding. PubMed DOI PMC

Knejzlik, Z. et al. The mycobacterial guaB1 gene encodes a guanosine 5’-monophosphate reductase with a cystathionine-beta-synthase domain. PubMed DOI PMC

Delumeau, O. et al. The dynamic protein partnership of RNA polymerase in PubMed DOI

Lin, W. et al. Structural basis of PubMed DOI PMC

Unger, T., Jacobovitch, Y., Dantes, A., Bernheim, R. & Peleg, Y. Applications of the restriction free (RF) cloning procedure for molecular manipulations and protein expression. PubMed DOI

Currinn, H., Guscott, B., Balklava, Z., Rothnie, A. & Wassmer, T. APP controls the formation of PI(3,5)P(2) vesicles through its binding of the PIKfyve complex. PubMed DOI PMC

Sikova, M. et al. Ms1 RNA increases the amount of RNA polymerase in PubMed DOI

Huff, J., Czyz, A., Landick, R. & Niederweis, M. Taking phage integration to the next level as a genetic tool for mycobacteria. PubMed DOI PMC

van Kessel, J. C. & Hatfull, G. F. Recombineering in PubMed DOI

Tropea, J. E., Cherry, S. & Waugh, D. S. Expression and purification of soluble His(6)-tagged TEV protease. PubMed DOI

Lin, T. I. & Morales, M. F. Application of a one-step procedure for measuring inorganic phosphate in the presence of proteins: the actomyosin ATPase system. PubMed DOI

Villanueva, R. A. M. & Chen, Z. J. ggplot2: Elegant Graphics for Data Analysis, 2nd edition.

Krasny, L. & Gourse, R. L. An alternative strategy for bacterial ribosome synthesis: PubMed DOI PMC

Qi, Y. & Hulett, F. M. PhoP-P and RNA polymerase sigmaA holoenzyme are sufficient for transcription of Pho regulon promoters in PubMed DOI

Kang, K. R. & Kim, Y. W. A simple protocol of DNA sequencing with 10% formamide for dissolving G/C compression. DOI

Perez-Riverol, Y. et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. PubMed DOI PMC

Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. PubMed DOI

Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. PubMed DOI PMC

Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. PubMed DOI

Bepler, T., Kelley, K., Noble, A. J. & Berger, B. Topaz-Denoise: general deep denoising models for cryoEM and cryoET. PubMed DOI PMC

Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. PubMed DOI PMC

Kimanius, D., Dong, L., Sharov, G., Nakane, T. & Scheres, S. H. W. New tools for automated cryo-EM single-particle analysis in RELION-4.0. PubMed DOI PMC

Burnley, T., Palmer, C. M. & Winn, M. Recent developments in the CCP-EM software suite. PubMed DOI PMC

Jakobi, A. J., Wilmanns, M. & Sachse, C. Model-based local density sharpening of cryo-EM maps. PubMed PMC

Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. PubMed DOI PMC

Naydenova, K. & Russo, C. J. Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy. PubMed DOI PMC

Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. PubMed DOI

Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. PubMed DOI PMC

Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. PubMed DOI

Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. PubMed DOI PMC

Meng, E. C. et al. UCSF ChimeraX: tools for structure building and analysis. PubMed DOI PMC

Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. PubMed DOI PMC

Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. PubMed DOI PMC

Cerny, J. et al. Structural alphabets for conformational analysis of nucleic acids available at dnatco.datmos.org. PubMed DOI PMC

Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. PubMed DOI

Winn, M. D. et al. Overview of the CCP4 suite and current developments. PubMed DOI PMC

Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. PubMed DOI

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PDB
8Q3I, 8QN8, 8QU6, 8R3M, 8R2M, 8R6P, 8R6R, 8QTI, 7PP4, 5TW1, 5VI5, 6EE8, 4XLS, 6YXU, 6YYS, 5VI8, 5UHC