Skip to main content
SpringerLink
Log in

Purification and characterization of recombinant Deinococcus radiodurans RNA Polymerase

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

Abstract

The radioresistant bacterium Deinococcus radiodurans is one of the most interesting models for studies of cell stress resistance. Analysis of the mechanisms of gene expression in D. radiodurans revealed some specific features of the transcription apparatus that might play a role in cell resistance to DNA-damaging conditions. In particular, RNA polymerase from D. radiodurans forms unstable promoter complexes and during transcription elongation has a much higher rate of RNA cleavage than RNA polymerase from Escherichia coli. Analysis of the structure and functions of D. radiodurans RNA polymerase is complicated due to the absence of convenient genetic systems for making mutations in the RNA polymerase genes and difficulties with enzyme purification. In this work, we developed a system for expression of D. radiodurans RNA polymerase in E. coli cells. We obtained an expression vector encoding all core RNA polymerase subunits and defined optimal conditions for the expression and purification of the RNA polymerase. It was found that D. radiodurans RNA polymerase has much higher rates of RNA cleavage than E. coli RNA polymerase under a wide range of conditions, including variations in the concentration of catalytic magnesium ions and pH values of the reaction buffer. The expression system can be used for further studies of the RNA cleavage reaction and the mechanisms of transcription regulation in D. radiodurans, including analysis of mutant RNA polymerase variants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

DTT:

dithiothreitol

EC:

elongation complex

IPTG:

isopropyl-β-D-1-thiogalactopyranoside

RNAP:

RNA polymerase

References

  1. Haugen, S. P., Ross, W., and Gourse, R. L. (2008) Advances in bacterial promoter recognition and its control by factors that do not bind DNA, Nat. Rev. Microbiol., 6, 507–519.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Nudler, E. (2009) RNA polymerase active center: the molecular engine of transcription, Annu. Rev. Biochem., 78, 335–361.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Pupov, D. V., and Kulbachinskiy, A. V. (2010) Structural dynamics of the active center of multisubunit RNA poly-merases during RNA synthesis and proofreading, Mol. Biol. (Moscow), 44, 573–590.

    Article  CAS  Google Scholar 

  4. Sydow, J. F., and Cramer, P. (2009) RNA polymerase fidelity and transcriptional proofreading, Curr. Opin. Struct. Biol., 19, 732–739.

    Article  CAS  PubMed  Google Scholar 

  5. Gordon, A. J., Halliday, J. A., Blankschien, M. D., Burns, P. A., Yatagai, F., and Herman, C. (2009) Transcriptional infidelity promotes heritable phenotypic change in a bistable gene network, PLoS Biol., 7, e44.

  6. Nudler, E. (2012) RNA polymerase backtracking in gene regulation and genome instability, Cel., 149, 1438–1445.

    Article  CAS  Google Scholar 

  7. Cheung, A. C., and Cramer, P. (2011) Structural basis of RNA polymerase II backtracking, arrest and reactivation, Natur., 471, 249–253.

    Article  CAS  Google Scholar 

  8. Sekine, S., Murayama, Y., Svetlov, V., Nudler, E., and Yokoyama, S. (2015) The ratcheted and ratchetable struc-tural states of RNA polymerase underlie multiple transcrip-tional functions, Mol. Cel., 57, 408–421.

    Article  CAS  Google Scholar 

  9. Sosunova, E., Sosunov, V., Epshtein, V., Nikiforov, V., and Mustaev, A. (2013) Control of transcriptional fidelity by active center tuning as derived from RNA polymerase endonuclease reaction, J. Biol. Chem., 288, 6688–6703.

    Article  CAS  PubMed  Google Scholar 

  10. Slade, D., and Radman, M. (2011) Oxidative stress resist-ance in Deinococcus radiodurans, Microbiol. Mol. Biol. Rev., 75, 133–191.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Liu, Y., Zhou, J., Omelchenko, M. V., Beliaev, A. S., Venkateswaran, A., Stair, J., Wu, L., Thompson, D. K., Xu, D., Rogozin, I. B., Gaidamakova, E. K., Zhai, M., Makarova, K. S., Koonin, E. V., and Daly, M. J. (2003) Transcriptome dynamics of Deinococcus radiodurans recov-ering from ionizing radiation, Proc. Natl. Acad. Sci. US., 100, 4191–4196.

    Article  CAS  Google Scholar 

  12. Luan, H., Meng, N., Fu, J., Chen, X., Xu, X., Feng, Q., Jiang, H., Dai, J., Yuan, X., Lu, Y., Roberts, A. A., Luo, X., Chen, M., Xu, S., Li, J., Hamilton, C. J., Fang, C., and Wang, J. (2014) Genome-wide transcriptome and antioxi-dant analyses on gamma-irradiated phases of deinococcus radiodurans R1, PloS One, 9, e85649.

  13. Kulbachinskiy, A., Bass, I., Bogdanova, E., Goldfarb, A., and Nikiforov, V. (2004) Cold sensitivity of thermophilic and mesophilic RNA polymerases, J. Bacteriol., 186, 7818–7820.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Barinova, N., Zhilina, E., Bass, I., Nikiforov, V., and Kulbachinskiy, A. (2008) Lineage-specific amino acid sub-stitutions in region 2 of the RNA polymerase sigma subunit affect the temperature of promoter opening, J. Bacteriol., 190, 3088–3092.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Miropolskaya, N., Ignatov, A., Bass, I., Zhilina, E., Pupov, D., and Kulbachinskiy, A. (2012) Distinct functions of regions 1.1 and 1.2 of RNA polymerase sigma subunits from Escherichia coli and Thermus aquaticus in transcription ini-tiation, J. Biol. Chem., 287, 23779–23789.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. 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 complex-es, Nucleic Acids Res., 40, 11352–11362.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Pupov, D. V., Barinova, N. A., and Kulbachinskiy, A. V. (2008) Analysis of RNA cleavage by RNA polymerases from Escherichia coli and Deinococcus radiodurans, Biochemistry (Moscow., 73, 725–729.

    Article  CAS  Google Scholar 

  18. Miropolskaya, N., Artsimovitch, I., Klimasauskas, S., Nikiforov, V., and Kulbachinskiy, A. (2009) Allosteric con-trol of catalysis by the F loop of RNA polymerase, Proc. Natl. Acad. Sci. US., 106, 18942–18947.

    Article  CAS  Google Scholar 

  19. Miropolskaya, N., Esyunina, D., Klimasauskas, S., Nikiforov, V., Artsimovitch, I., and Kulbachinskiy, A. (2014) Interplay between the trigger loop and the F loop during RNA polymerase catalysis, Nucleic Acids Res., 42, 544–552.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Svetlov, V., and Artsimovitch, I. (2015) Purification of bac-terial RNA polymerase: tools and protocols, Methods Mol. Biol., 1276, 13–29.

    Article  PubMed Central  PubMed  Google Scholar 

  21. Kuznedelov, K., Minakhin, L., and Severinov, K. (2003) Preparation and characterization of recombinant Thermus aquaticus RNA polymerase, Methods Enzymol., 370, 94–108.

    Article  CAS  PubMed  Google Scholar 

  22. Yang, X., and Lewis, P. J. (2008) Overproduction and purification of recombinant Bacillus subtilis RNA poly-merase, Protein Expr. Purif., 59, 86–93.

    Article  CAS  PubMed  Google Scholar 

  23. Banerjee, R., Rudra, P., Prajapati, R. K., Sengupta, S., and Mukhopadhyay, J. (2014) Optimization of recombinant Mycobacterium tuberculosis RNA polymerase expression and purification, Tuberculosi., 94, 397–404.

    Article  CAS  Google Scholar 

  24. Hu, Y., Morichaud, Z., Perumal, A. S., Roquet-Baneres, F., and Brodolin, K. (2014) Mycobacterium RbpA cooper-ates with the stress-response sigmaB subunit of RNA poly-merase in promoter DNA unwinding, Nucleic Acids Res., 42, 10399–10408.

    Article  PubMed Central  PubMed  Google Scholar 

  25. Cheng, C. Y., Yu, Y. J., and Yang, M. T. (2010) Coexpression of omega subunit in E. coli is required for the maintenance of enzymatic activity of Xanthomonas campestris pv. campestris RNA polymerase, Protein Expr. Purif., 69, 91–98.

    Article  CAS  PubMed  Google Scholar 

  26. Sosunov, V., Zorov, S., Sosunova, E., Nikolaev, A., Zakeyeva, I., Bass, I., Goldfarb, A., Nikiforov, V., Severinov, K., and Mustaev, A. (2005) The involvement of the aspartate triad of the active center in all catalytic activ-ities of multisubunit RNA polymerase, Nucleic Acids Res., 33, 4202–4211.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Zenkin, N., Yuzenkova, Y., and Severinov, K. (2006) Transcript-assisted transcriptional proofreading, Scienc., 313, 518–520.

    Article  CAS  Google Scholar 

  28. Laptenko, O., Lee, J., Lomakin, I., and Borukhov, S. (2003) Transcript cleavage factors GreA and GreB act as transient catalytic components of RNA polymerase, EMBO J., 22, 6322–6334.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Sosunova, E., Sosunov, V., Kozlov, M., Nikiforov, V., Goldfarb, A., and Mustaev, A. (2003) Donation of catalyt-ic residues to RNA polymerase active center by transcrip-tion factor Gre, Proc. Natl. Acad. Sci. US., 100, 15469–15474.

    Article  CAS  Google Scholar 

  30. Orlova, M., Newlands, J., Das, A., Goldfarb, A., and Borukhov, S. (1995) Intrinsic transcript cleavage activity of RNA polymerase, Proc. Natl. Acad. Sci. US., 92, 4596–4600.

    Article  CAS  Google Scholar 

  31. Sosunov, V., Sosunova, E., Mustaev, A., Bass, I., Nikiforov, V., and Goldfarb, A. (2003) Unified two-metal mechanism of RNA synthesis and degradation by RNA polymerase, EMBO J., 22, 2234–2244.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Yuzenkova, Y., and Zenkin, N. (2010) Central role of the RNA polymerase trigger loop in intrinsic RNA hydrolysis, Proc. Natl. Acad. Sci. US., 107, 10878–10883.

    Article  CAS  Google Scholar 

  33. McGlynn, P., Savery, N. J., and Dillingham, M. S. (2012) The conflict between DNA replication and transcription, Mol. Microbiol., 85, 12–20.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

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

    D. M. Esyunina & A. V. Kulbachinskiy

Authors
  1. D. M. Esyunina
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. V. Kulbachinskiy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to D. M. Esyunina or A. V. Kulbachinskiy.

Additional information

Published in Russian in Biokhimiya, 2015, Vol. 80, No. 10, pp. 1542-1550.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM15-118, August 9, 2015.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Esyunina, D.M., Kulbachinskiy, A.V. Purification and characterization of recombinant Deinococcus radiodurans RNA Polymerase. Biochemistry Moscow 80, 1271–1279 (2015). https://doi.org/10.1134/S0006297915100077

Download citation

  • Received:

  • Revised:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297915100077

Keywords

Search

Navigation