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Role of Interactions of the CRE Region of Escherichia coli RNA Polymerase with Nontemplate DNA during Promoter Escape

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Abstract

RNA polymerase (RNAP) recognizes promoter DNA through many interactions that determine specificity of transcription initiation. In addition to the dedicated transcription initiation σ factor in bacteria, the core enzyme of RNAP can also participate in promoter recognition. In particular, guanine residue at the +2 position (+2G) of the nontemplate DNA strand is bound in the CRE pocket formed by the RNAP β subunit. Here, we analyzed the role of these contacts in the process of promoter escape by RNAP by studying point mutations in the β subunit of Escherichia coli RNAP that disrupted these interactions. We found that the presence of +2G in the promoter slowed down the rate of promoter escape and increased proportion of inactive complexes. Amino acid substitutions in the CRE pocket decreased the promoter complex stability and changed the pattern of short RNA products synthesized during initiation, but did not significantly affect the rate of transition to elongation, regardless of the presence of +2G. Thus, the contacts of the CRE pocket with +2G do not make a significant contribution to the kinetics of promoter escape by RNAP, while the observed changes in the efficiency of abortive synthesis are not directly related to the rate of promoter escape.

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Abbreviations

CRE:

core recognition element

ITS:

initially transcribed sequence

RNAP:

RNA polymerase

RPint:

initiating RNAP-promoter complex

RPo:

open RNAP-promoter complex

WT:

wild type

REFERENCES

  1. Feklistov, A., Sharon, B. D., Darst, S. A., and Gross, C. A. (2014) Bacterial sigma factors: a historical, structural, and genomic perspective, Annu. Rev. Microbiol., 68, 357-376, doi: https://doi.org/10.1146/annurev-micro-092412-155737 .

    Article  CAS  PubMed  Google Scholar 

  2. Zhang, Y., Feng, Y., Chatterjee, S., Tuske, S., Ho, M. X., Arnold, E., and Ebright, R. H. (2012) Structural basis of transcription initiation, Science, 338, 1076-1080, doi: https://doi.org/10.1126/science.1227786 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Basu, R. S., Warner, B. A., Molodtsov, V., Pupov, D., Esyunina, D., Fernandez-Tornero, C., Kulbachinskiy, A., and Murakami, K. S. (2014) Structural basis of transcription initiation by bacterial RNA polymerase holoenzyme, J. Biol. Chem., 289, 24549-24559, doi: https://doi.org/10.1074/jbc.M114.584037 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zuo, Y., and Steitz, T. A. (2015) Crystal structures of the E. coli transcription initiation complexes with a complete bubble, Mol. Cell, 58, 534-540, doi: https://doi.org/10.1016/j.molcel.2015.03.010 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Mitchell, J. E., Zheng, D., Busby, S. J., and Minchin, S. D. (2003) Identification and analysis of “extended –10” promoters in Escherichia coli, Nucleic Acids Res., 31, 4689-4695, doi: https://doi.org/10.1093/nar/gkg694 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Vvedenskaya, I. O., Vahedian-Movahed, H., Zhang, Y., Taylor, D. M., Ebright, R. H., and Nickels, B. E. (2016) Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection, Proc. Natl. Acad. Sci. USA, 113, E2899-2905, doi: https://doi.org/10.1073/pnas.1603271113 .

    Article  CAS  PubMed  Google Scholar 

  7. Kulbachinskiy, A., and Mustaev, A. (2006) Region 3.2 of the sigma subunit contributes to the binding of the 3′-initiating nucleotide in the RNA polymerase active center and facilitates promoter clearance during initiation, J. Biol. Chem., 281, 18273-18276, doi: https://doi.org/10.1074/jbc.C600060200 .

    Article  CAS  PubMed  Google Scholar 

  8. Pupov, D., Kuzin, I., Bass, I., and Kulbachinskiy, A. (2014) Distinct functions of the RNA polymerase sigma subunit region 3.2 in RNA priming and promoter escape, Nucleic Acids Res., 42, 4494-4504, doi: https://doi.org/10.1093/nar/gkt1384 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Duchi, D., Bauer, D. L., Fernandez, L., Evans, G., Robb, N., Hwang, L. C., Gryte, K., Tomescu, A., Zawadzki, P., Morichaud, Z., Brodolin, K., and Kapanidis, A. N. (2016) RNA polymerase pausing during initial transcription, Mol. Cell, 63, 939-950, doi: https://doi.org/10.1016/j.molcel.2016.08.011 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Dulin, D., Bauer, D. L. V., Malinen, A. M., Bakermans, J. J. W., Kaller, M., Morichaud, Z., Petushkov, I., Depken, M., Brodolin, K., Kulbachinskiy, A., and Kapanidis, A. N. (2018) Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria, Nat. Commun., 9, 1478, doi: https://doi.org/10.1038/s41467-018-03902-9 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Vvedenskaya, I. O., Vahedian-Movahed, H., Bird, J. G., Knoblauch, J. G., Goldman, S. R., Zhang, Y., Ebright, R. H., and Nickels, B. E. (2014) Transcription. Interactions between RNA polymerase and the “core recognition element” counteract pausing, Science, 344, 1285-1289, doi: https://doi.org/10.1126/science.1253458 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Petushkov, I., Pupov, D., Bass, I., and Kulbachinskiy, A. (2015) Mutations in the CRE pocket of bacterial RNA polymerase affect multiple steps of transcription, Nucleic Acids Res., 43, 5798-5809, doi: https://doi.org/10.1093/nar/gkv504 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wiesler, S. C., Weinzierl, R. O., and Buck, M. (2013) An aromatic residue switch in enhancer-dependent bacterial RNA polymerase controls transcription intermediate complex activity, Nucleic Acids Res., 41, 5874-5886, doi: https://doi.org/10.1093/nar/gkt271 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Mekler, V., Kortkhonjia, E., Mukhopadhyay, J., Knight, J., Revyakin, A., Kapanidis, A. N., Niu, W., Ebright, Y. W., Levy, R., and Ebright, R. H. (2002) Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex, Cell, 108, 599-614.

    Article  CAS  Google Scholar 

  15. Straney, D. C., and Crothers, D. M. (1987) A stressed intermediate in the formation of stably initiated RNA chains at the Escherichia colilacUV5 promoter, J. Mol. Biol., 193, 267-278, doi: https://doi.org/10.1016/0022-2836(87)90218-x .

    Article  CAS  PubMed  Google Scholar 

  16. Kapanidis, A. N., Margeat, E., Ho, S. O., Kortkhonjia, E., Weiss, S., and Ebright, R. H. (2006) Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism, Science, 314, 1144-1147, doi: https://doi.org/10.1126/science.1131399 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Revyakin, A., Liu, C., Ebright, R. H., and Strick, T. R. (2006) Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching, Science, 314, 1139-1143, doi: https://doi.org/10.1126/science.1131398 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Henderson, K. L., Felth, L. C., Molzahn, C. M., Shkel, I., Wang, S., Chhabra, M., Ruff, E. F., Bieter, L., Kraft, J. E., and Record, M. T., Jr. (2017) Mechanism of transcription initiation and promoter escape by E. coli RNA polymerase, Proc. Natl. Acad. Sci. USA, 114, E3032-E3040, doi: https://doi.org/10.1073/pnas.1618675114 .

    Article  CAS  PubMed  Google Scholar 

  19. Winkelman, J. T., and Gourse, R. L. (2017) Open complex DNA scrunching: a key to transcription start site selection and promoter escape, BioEssays, 39, doi: https://doi.org/10.1002/bies.201600193 .

    Article  Google Scholar 

  20. Carpousis, A. J., and Gralla, J. D. (1985) Interaction of RNA polymerase with lacUV5 promoter DNA during mRNA initiation and elongation. Footprinting, methylation, and rifampicin-sensitivity changes accompanying transcription initiation, J. Mol. Biol., 183, 165-177, doi: https://doi.org/10.1016/0022-2836(85)90210-4 .

    Article  CAS  PubMed  Google Scholar 

  21. Ko, J., and Heyduk, T. (2014) Kinetics of promoter escape by bacterial RNA polymerase: effects of promoter contacts and transcription bubble collapse, Biochem. J., 463, 135-144, doi: https://doi.org/10.1042/BJ20140179 .

    Article  CAS  PubMed  Google Scholar 

  22. Samanta, S., and Martin, C. T. (2013) Insights into the mechanism of initial transcription in Escherichia coli RNA polymerase, J. Biol. Chem., 288, 31993-32003, doi: https://doi.org/10.1074/jbc.M113.497669 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Murakami, K. S., Masuda, S., and Darst, S. A. (2002) Structural basis of transcription initiation: RNA polymerase holoenzyme at 4 Å resolution, Science, 296, 1280-1284, doi: https://doi.org/10.1126/science.1069594 .

    Article  CAS  PubMed  Google Scholar 

  24. Li, L., Molodtsov, V., Lin, W., Ebright, R. H., and Zhang, Y. (2020) RNA extension drives a stepwise displacement of an initiation-factor structural module in initial transcription, Proc. Natl. Acad. Sci. USA, 117, 5801-5809, doi: https://doi.org/10.1073/pnas.1920747117 .

    Article  CAS  PubMed  Google Scholar 

  25. Petushkov, I., Esyunina, D., Mekler, V., Severinov, K., Pupov, D., and Kulbachinskiy, A. (2017) Interplay between sigma region 3.2 and secondary channel factors during promoter escape by bacterial RNA polymerase, Biochem. J., 474, 4053-4064, doi: https://doi.org/10.1042/BCJ20170436 .

    Article  CAS  PubMed  Google Scholar 

  26. Kammerer, W., Deuschle, U., Gentz, R., and Bujard, H. (1986) Functional dissection of Escherichia coli promoters: information in the transcribed region is involved in late steps of the overall process, EMBO J., 5, 2995-3000.

    Article  CAS  Google Scholar 

  27. Hsu, L. M., Cobb, I. M., Ozmore, J. R., Khoo, M., Nahm, G., Xia, L., Bao, Y., and Ahn, C. (2006) Initial transcribed sequence mutations specifically affect promoter escape properties, Biochemistry, 45, 8841-8854, doi: https://doi.org/10.1021/bi060247u .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hsu, L. M. (2002) Promoter clearance and escape in prokaryotes, Biochim. Biophys. Acta, 1577, 191-207, doi: https://doi.org/10.1016/s0167-4781(02)00452-9 .

    Article  CAS  PubMed  Google Scholar 

  29. Hsu, L. M. (2009) Monitoring abortive initiation, Methods, 47, 25-36, doi: https://doi.org/10.1016/j.ymeth.2008.10.010 .

    Article  CAS  PubMed  Google Scholar 

  30. Vo, N. V., Hsu, L. M., Kane, C. M., and Chamberlin, M. J. (2003) In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 3. Influences of individual DNA elements within the promoter recognition region on abortive initiation and promoter escape, Biochemistry, 42, 3798-3811, doi: https://doi.org/10.1021/bi026962v .

    Article  CAS  PubMed  Google Scholar 

  31. Heyduk, E., and Heyduk, T. (2018) DNA template sequence control of bacterial RNA polymerase escape from the promoter, Nucleic Acids Res., 46, 4469-4486, doi: https://doi.org/10.1093/nar/gky172 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lerner, E., Chung, S., Allen, B. L., Wang, S., Lee, J., Lu, S. W., Grimaud, L. W., Ingargiola, A., Michalet, X., Alhadid, Y., Borukhov, S., Strick, T. R., Taatjes, D. J., and Weiss, S. (2016) Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase, Proc. Natl. Acad. Sci. USA, 113, E6562-E6571, doi: https://doi.org/10.1073/pnas.1605038113 .

    Article  CAS  PubMed  Google Scholar 

  33. Mekler, V., Pavlova, O., and Severinov, K. (2011) The interaction of E. coli RNA polymerase σ70 subunit with promoter elements in the context of free σ70, RNA polymerase holoenzyme and the β′–σ70 complex, J. Biol. Chem., 286, 270-279, doi: https://doi.org/10.1074/jbc.M110.174102 .

    Article  CAS  PubMed  Google Scholar 

  34. Mekler, V., and Severinov, K. (2015) Use of RNA polymerase molecular beacon assay to measure RNA polymerase interactions with model promoter fragments, Methods Mol. Biol., 1276, 199-210, doi: https://doi.org/10.1007/978-1-4939-2392-2_11 .

    Article  CAS  PubMed  Google Scholar 

  35. Svetlov, V., and Artsimovitch, I. (2015) Purification of bacterial RNA polymerase: tools and protocols, Methods Mol. Biol., 1276, 13-29, doi: https://doi.org/10.1007/978-1-4939-2392-2_2 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Petushkov, I., Esyunina, D., and Kulbachinskiy, A. (2017) σ38-Dependent promoter-proximal pausing by bacterial RNA polymerase, Nucleic Acids Res., 45, 3006-3016, doi: https://doi.org/10.1093/nar/gkw1213 .

    Article  CAS  PubMed  Google Scholar 

  37. Pupov, D., Petushkov, I., Esyunina, D., Murakami, K. S., and Kulbachinskiy, A. (2018) Region 3.2 of the σ factor controls the stability of rRNA promoter complexes and potentiates their repression by DksA, Nucleic Acids Res., 46, 11477-11487, doi: https://doi.org/10.1093/nar/gky919 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Pupov, D., Esyunina, D., Feklistov, A., and Kulbachinskiy, A. (2013) Single-strand promoter traps for bacterial RNA polymerase, Biochem. J., 452, 241-248, doi: https://doi.org/10.1042/BJ20130069 .

    Article  CAS  PubMed  Google Scholar 

  39. Pupov, D., Miropolskaya, N., Sevostyanova, A., Bass, I., Artsimovitch, I., and Kulbachinskiy, A. (2010) Multiple roles of the RNA polymerase β′ SW2 region in transcription initiation, promoter escape, and RNA elongation, Nucleic Acids Res., 38, 5784-5796, doi: https://doi.org/10.1093/nar/gkq355 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Mekler, V., Minakhin, L., Kuznedelov, K., Mukhamedyarov, D., and Severinov, K. (2012) RNA polymerase–promoter interactions determining different stability of the Escherichia coli and Thermus aquaticus transcription initiation complexes, Nucleic Acids Res., 40, 11352-11362, doi: https://doi.org/10.1093/nar/gks973 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Mekler, V., Minakhin, L., and Severinov, K. (2011) A critical role of downstream RNA polymerase–promoter interactions in the formation of initiation complex, J. Biol. Chem., 286, 22600-22608, doi: https://doi.org/10.1074/jbc.M111.247080 .

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Sugimoto, N., Nakano, S., Katoh, M., Matsumura, A., Nakamuta, H., Ohmichi, T., Yoneyama, M., and Sasaki, M. (1995) Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes, Biochemistry, 34, 11211-11216, doi: https://doi.org/10.1021/bi00035a029 .

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors are grateful to Dr. Irina Artsimovich for providing plasmids.

Funding

This work was supported by the Russian Science Foundation (project No. 17-14-01393).

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  1. Institute of Molecular Genetics, Russian Academy of Sciences, 123182, Moscow, Russia

    I. V. Petushkov & A. V. Kulbachinskiy

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  1. I. V. Petushkov
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Correspondence to I. V. Petushkov.

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The authors declare no conflict of interest in financial or any other sphere. This article does not contain any studies with human participants or animals performed by any of the authors.

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Petushkov, I., Kulbachinskiy, A. Role of Interactions of the CRE Region of Escherichia coli RNA Polymerase with Nontemplate DNA during Promoter Escape. Biochemistry Moscow 85, 792–800 (2020). https://doi.org/10.1134/S000629792007007X

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