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Mycobacterial Held Is a Nucleic Acids-Clearing Factor for RNA

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Mycobacterial Held Is a Nucleic Acids-Clearing Factor for RNA bioRxiv preprint doi: https://doi.org/10.1101/2020.07.20.211821; this version posted July 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Mycobacterial HelD is a nucleic acids-clearing factor for RNA 2 polymerase 3 4 5 Tomáš Koubaa*#, Tomáš Koval’b*, Petra Sudzinovác*, Jiří Pospíšilc, Barbora Brezovskác, Jarmila 6 Hnilicovác, Hana Šanderovác, Martina Janouškovác, Michaela Šikovác, Petr Haladac, Michal 7 Sýkorad, Ivan Barvíke, Jiří Nováčekf, Mária Trundováb, Jarmila Duškováb, Tereza Skálováb, 8 URee Chong, Katsuhiko S. Murakamig, Jan Dohnálekb#, Libor Krásnýc# 9 10 a EMBL Grenoble, 71 Avenue des Martyrs, France 11 b Institute of Biotechnology of the Czech Academy of Sciences, Centre BIOCEV, Prumyslova 12 595, 25250 Vestec, Czech Republic 13 c Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic 14 d Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic 15 e Charles University, Faculty of Mathematics and Physics, Institute of Physics, Prague, Czech 16 Republic 17 f CEITEC, Masaryk University, Brno, Czech Republic 18 g Department of Biochemistry and Molecular Biology, The Center for RNA Molecular Biology, 19 Pennsylvania State University, University Park, PA 16802, USA 20 21 22 23 24 25 *Equal contribution 26 #Corresponding author: [email protected], [email protected], [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.20.211821; this version posted July 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 27 SUMMARY 28 RNA synthesis is central to life, and RNA polymerase depends on accessory factors for 29 recovery from stalled states and adaption to environmental changes. Here we investigated 30 the mechanism by which a helicase-like factor HelD recycles RNA polymerase. We report a 31 cryo-EM structure of an unprecedented complex between the Mycobacterium smegmatis 32 RNA polymerase and HelD. The crescent-shaped HelD simultaneously penetrates deep into 33 two RNA polymerase channels that are responsible for DNA binding and substrate delivery 34 to the active site, thereby locking RNA polymerase in an inactive state. We show that HelD 35 prevents non-specific interactions between RNA polymerase and DNA and dissociates 36 transcription elongation complexes, but does not inhibit RNA polymerase binding to the 37 initiation σ factor. The liberated RNA polymerase can either stay dormant, sequestered by 38 HelD, or upon HelD release, restart transcription. Our results provide insights into the 39 architecture and regulation of the highly medically-relevant mycobacterial transcription 40 machinery and define HelD as a clearing factor that removes undesirable nucleic acids from 41 RNA polymerase. 42 A smoothly functioning transcription machinery is essential for maintaining the 43 physiologically relevant levels of gene products and adequate changes in transcription are 44 necessary for cell survival when the environment changes. In bacteria, transcription is 45 executed by a single enzyme, DNA-dependent RNA polymerase [RNAP; composition of the 1 46 core enzyme: α2ββ’ω ]. The RNAP core is capable of transcription elongation and termination 47 but not initiation. To initiate, a factor is required to form a holoenzyme that recognizes 48 specific DNA sequences, promoters2. Besides the core subunits that are conserved in all 49 bacteria some species contain additional subunits/associated proteins, such as δ and ε that 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.20.211821; this version posted July 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 50 are present in Firmicutes3,4, HelD that is present in Gram-positive bacteria5, or CarD and RbpA6 51 that are found in Actinobacteria. The smooth functioning of the transcription machinery then 52 depends on concerted activities of RNAP and numerous transcription factors. 53 One of these factors is HelD5, a protein similar to SF1 helicases7 that associates with 54 the RNAP core in the model gram-positive bacterium Bacillus subtilis (Bsu) where it was 55 shown to be involved in transcriptional recycling8. HelD binds and hydrolyzes ATP and this is 56 accompanied by conformational changes in the protein as demonstrated by SAXS 57 experiments9. The absence of HelD from Bsu cells results in prolonged lag phase during 58 outgrowth of stationary phase cells when diluted into fresh medium; overexpression of HelD 59 then accelerates spore formation10. However, the structure of HelD, its binding mode to 60 RNAP, and mechanistic details of its function are unknown. 61 Here, we present structural data for HelD from Mycobacterium smegmatis (Msm) in 62 complex with the RNAP core and provide insights into its function. We solved the 3D structure 63 of three complexes of Msm RNAP and HelD by cryogenic electron microscopy (cryo-EM). The 64 structures represent a so far unknown type of interaction between an RNAP and a protein. 65 Furthermore, we show that mycobacterial HelD and σA may simultaneously bind to the RNAP 66 core and we provide biochemical evidence showing that in addition to ATP, HelD can also 67 hydrolyze GTP. Finally, we demonstrate that HelD can both prevent binding of the RNAP core 68 to non-specific DNA and actively remove RNAP from stalled elongation complexes. 69 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.20.211821; this version posted July 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 70 Results 71 Cryo-EM of Msm RNAP-HelD complex 72 Our long-term attempts to crystalize Bsu HelD, RNAP core, or their complex failed; our cryo- 73 EM experiments with the Bsu RNAP core were not successful; also, our recent SAXS-based 74 data for the Bsu HelD-RNAP complex were not fully conclusive. However, in co- 75 immunoprecipitation experiments with Msm RNAP we identified MSMEG_2174, a potential 76 homolog of Bsu HelD (Figure S1). We also solved the X-ray crystal structure of Bsu HelD C- 77 terminal domain (CTD), which was then used as a guide for building the model of Msm HelD. 78 We reconstituted a complex of Msm RNAP core and Msm HelD from purified 79 recombinant proteins (Figure S2), and froze an isolated homogenous fraction of the complex 80 on cryo-EM grids. We collected multiple preliminary cryo-EM datasets, which allowed us to 81 optimize the cryo-EM conditions for high-resolution three-dimensional (3D) single-particle 82 reconstructions (Figure S3). We identified two major 3D classes (State I and State II, Figure 83 S4) at overall resolution ~3.1 Å (plus one subclass at ~3.6 Å), visualising almost the complete 84 structure of HelD bound to the RNAP core in two conformations (Figure 1a,b, Movies 1,2), 85 and one minor class (State III; Figure S4), at ~3.5 Å, which delineates only two domains of HelD 86 binding to the RNAP core (Figure 1c, Movie 3). The structures of States I and II share the same 87 overall fold of HelD, with a crescent-like shape. The main body of the crescent is sitting in 88 between the domain 2 of the RNAP β subunit (also called β-lobe domain) and the funnel and 89 cleft/jaw of the β' subunit, burying about 774 and 2608 Å211 in State I and 1490 and 3623 Å2 90 in State II of binding surface area of β and β' subunits, respectively. One end of the crescent 91 protrudes deep into the primary channel, and the other end into the secondary channel of 92 the RNAP core. Indeed, to be able to reach both RNAP channels simultaneously, the HelD 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.20.211821; this version posted July 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 93 protein is markedly elongated, around 200 Å along the outer edge of the virtual crescent, and 94 the two ends of the HelD protein are separated by ~75 Å (State II; Figure 1b). 95 The HelD protein itself is divided into six structured domains (Figure 1d-g), several of 96 which possess unique novel folds. Interestingly, the 1A domain is composed of two parts (1A- 97 1 and 1A-2) that are separated in the primary amino acid sequence by the intervening HelD- 98 specific domain. According to the position of the HelD domains within the primary channel 99 (PCh) and active site (AS), we name State I: PCh-engaged, State II: PCh-engaged and AS- 100 interfering, and State III: PCh dis-engaged and AS-interfering (Figure 1a-c). 101 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.20.211821; this version posted July 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 102 103 Figure 1: Cryo-EM structures of Msm HelD-RNAP complexes. 104 a, b and c, Atomic model surface representation of three identified Msm HelD-RNAP 105 complexes: State I – PCh-engaged, State II – PCh-engaged AS-interfering and State III – PCh- 106 dis-engaged AS-interfering. When fully ordered in State I and II (a and b), the HelD protein 107 (color coded as in d) forms a crescent-like shape, ends of which are protruding to the primary 108 and secondary channel of the RNAP core.
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