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Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo

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Abstract

5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

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References

  1. Harms, J., Schluenzen, F., Zarivach, R., Bashan, A., Gat, S., Agmon, I., Bartels, H., Franceschi, F., and Yonath, A. (2001) High resolution structure of the large ribosomal subunit from a mesophilic eubacterium, Cell, 107, 679–688.

    Article  CAS  PubMed  Google Scholar 

  2. Schuwirth, B. S., Borovinskaya, M. A., Hau, C. W., Zhang, W., Vila-Sanjurjo, A., Holton, J. M., and Cate, J. H. D. (2005) Structures of the bacterial ribosome at 3.5 resolution, Science, 310, 827–834.

    Article  CAS  PubMed  Google Scholar 

  3. Selmer, M., Dunham, C. M., Murphy, IV, F. V., Weixlbaumer, A., Petry, S., Kelley, A. C., Weir, J. R., and Ramakrishnan, V. (2006) Structure of the 70S ribosome complexed with mRNA and tRNA, Science, 313, 1935–1942.

    Article  CAS  PubMed  Google Scholar 

  4. Korostelev, A., Trakhanov, S., Laurberg, M., and Noller, H. F. (2006) Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements, Cell, 126, 1065–1077.

    Article  CAS  PubMed  Google Scholar 

  5. Yusupova, G., Jenner, L., Rees, B., Moras, D., and Yusupov, M. (2006) Structural basis for messenger RNA movement on the ribosome, Nature, 444, 391–394.

    Article  CAS  PubMed  Google Scholar 

  6. Voorhees, R. M., Weixlbaumer, A., Loakes, D., Kelley, A. C., and Ramakrishnan, V. (2009) Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome, Nat. Struct. Mol. Biol., 16, 528–533.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Schmeing, T. M., Voorhees, R. M., Kelley, A. C., Gao, Y. G., Murphy, IV, F. V., Weir, J. R., and Ramakrishnan, V. (2009) The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA, Science, 326, 688–694.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Gao, Y. G., Selmer, M., Dunham, C. M., Weixlbaumer, A., Kelley, A. C., and Ramakrishnan, V. (2009) The structure of the ribosome with elongation factor G trapped in the posttranslocational state, Science, 326, 694–699.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Lecompte, O., Ripp, R., Thierry, J. C., Moras, D., and Poch, O. (2002) Comparative analysis of ribosomal proteins in complete genomes: an example of reductive evolution at the domain scale, Nucleic Acids Res., 30, 5382–5390.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Ban, N., Beckmann, R., Cate, J. H., Dinman, J. D., Dragon, F., Ellis, S. R., Lafontaine, D. L., Lindahl, L., Liljas, A., Lipton, J. M., McAlear, M. A., Moore, P. B., Noller, H. F., Ortega, J., Panse, V. G., Ramakrishnan, V., Spahn, C. M., Steitz, T. A., Tchorzewski, M., Tollervey, D., Warren, A. J., Williamson, J. R., Wilson, D., Yonath, A., and Yusupov, M. (2014) A new system for naming ribosomal proteins, Curr. Opin. Stuct. Biol., 24, 1–5.

    Google Scholar 

  11. Lotti, M., Noah, M., Stoffler-Meilicke, M., and Stoffler, G. (1989) Localization of proteins L4, L5, L20 and L25 on the ribosomal surface by immunoelectron microscopy, Mol. Gen. Genet., 216, 245–253.

    Article  CAS  PubMed  Google Scholar 

  12. Douthwaite, S., Garrett, R. A., Wagner, R., and Feunteun, J. (1979) A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25, Nucleic Acids Res., 6, 2453–2470.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Shpanchenko, O. V., Zvereva, M. I., Dontsova, O. A., Nierhaus, K. H., and Bogdanov, A. A. (1996) 5S rRNA sugar-phosphate backbone protection in complexes with specific ribosomal proteins, FEBS Lett., 394, 71–75.

    Article  CAS  PubMed  Google Scholar 

  14. Gongadze, G. M., Meshcheryakov, V. A., Serganov, A. A., Fomenkova, N. P., Mudrik, E. S., Jonsson, B. H., Liljas, A., Nikonov, S. V., and Garber, M. B. (1999) N-terminal domain, residues 1–91, of ribosomal protein TL5 from Thermus thermophilus binds specifically and strongly to the region of 5S rRNA containing loop E, FEBS Lett., 451, 51–55.

    Article  CAS  PubMed  Google Scholar 

  15. Stoldt, M., Wohnert, J., Ohlenschlager, O., Gorlach, M., and Brown, L. R. (1999) The NMR structure of the 5S rRNA E-domain-protein L25 complex shows preformed and induced recognition, EMBO J., 18, 6508–6521.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Lu, M., and Steitz, T. A. (2000) Structure of Escherichia coli ribosomal protein L25 complexed with a 5S rRNA fragment at 1.8 resolution, Proc. Natl. Acad. Sci. USA, 97, 2023–2028.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Fedorov, R., Meshcheryakov, V., Gongadze, G., Fomenkova, N., Nevskaya, N., Selmer, M., Laurberg, M., Kristensen, O., Al-Karadaghi, S., Liljas, A., Garber, M., and Nikonov, S. (2001) Structure of ribosomal protein TL5 complexed with RNA provides new insights into the CTC family of stress proteins, Acta Crystallogr. Sect. D, 57, 968–976.

    Article  CAS  Google Scholar 

  18. Korepanov, A. P., Gongadze, G. M., and Garber, M. B. (2004) General stress protein CTC from Bacillus subtilis specifically binds to ribosomal 5S RNA, Biochemistry (Moscow), 69, 607–611.

    Article  CAS  Google Scholar 

  19. Korobeinikova, A. V., Gongadze, G. M., Korepanov, A. P., Eliseev, B. D., Bazhenova, M. V., and Garber, M. B. (2008) 5S rRNA-recognition module of CTC family proteins and its evolution, Biochemistry (Moscow), 73, 156–163.

    Article  CAS  Google Scholar 

  20. Spierer, P., Bogdanov, A. A., and Zimmermann, R. A. (1978) Parameters for interaction of ribosomal proteins L5, L18, and L25 with 5S RNA from Escherichia coli, Biochemistry, 17, 5394–5398.

    Article  CAS  PubMed  Google Scholar 

  21. Gongadze, G. M., Korepanov, A. P., Stolboushkina, E. A., Zelinskaya, N. V., Korobeinikova, A. V., Ruzanov, M. V., Eliseev, B. D., Nikonov, O. S., Nikonov, S. V., Garber, M. B., and Lim, V. I. (2005) The crucial role of conserved intermolecular H-bonds inaccessible to the solvent in formation and stabilization of the TL5-5S rRNA complex, J. Biol. Chem., 280, 16151–16156.

    Article  CAS  PubMed  Google Scholar 

  22. Korepanov, A. P., Korobeinikova, A. V., Shestakov, S. A., Garber, M. B., and Gongadze, G. M. (2012) Protein L5 is crucial for in vivo assembly of the bacterial 50S ribosomal subunit central protuberance, Nucleic Acids Res., 40, 9153–9159.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Hecker, M., and Volker, U. (1990) General stress proteins in Bacillus subtilis, FEMS Microbiol. Ecol., 74, 197–214.

    Article  CAS  Google Scholar 

  24. Schmalisch, M., Langbein, I., and Stulke, J. (2002) The general stress protein CTC of Bacillus subtilis is a ribosomal protein, J. Mol. Microbiol. Biotechnol., 4, 495–501.

    CAS  PubMed  Google Scholar 

  25. Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  26. Korepanov, A. P., Gongadze, G. M., Garber, M. B., Court, D. L., and Bubunenko, M. G. (2007) Importance of the 5S rRNA-binding ribosomal proteins for cell viability and translation in Escherichia coli, J. Mol. Biol., 366, 1199–1208.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Yu, D., Ellis, H. M., Lee, E. C., Jenkins, N. A., Copeland, N. G., and Court, D. L. (2000) An efficient recombination system for chromosome engineering in Escherichia coli, Proc. Natl. Acad. Sci. USA, 97, 5978–5983.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., Heyneker, H. L., Boyer, H. W., Crosa, J. H., and Falkow, S. (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system, Gene, 2, 95–113.

    Article  CAS  PubMed  Google Scholar 

  29. Bachmann, B. J. (1972) Pedigrees of some mutant strains of Escherichia coli K-12, Bacteriol. Rev., 36, 525–557.

    CAS  PubMed Central  PubMed  Google Scholar 

  30. Thomason, L. C., Costantino, N., and Court, D. L. (2007) Escherichia coli genome manipulation by P1 transduction, Curr. Protoc. Mol. Biol., 79, pp. 1.17.1–1.17.8, John Wiley & Sons, Inc.

    Article  Google Scholar 

  31. Erbe, R. W., Nau, M. M., and Leder, P. (1969) Translation and translocation of defined RNA messengers, J. Mol. Biol., 38, 441–460.

    Article  Google Scholar 

  32. Staehelin, T., Maglott, D. M., and Monro, R. E. (1969) On the catalytic center of peptidyl transfer: a part of the 50S ribosome structure, Cold Spring Harb. Symp. Quant. Biol., 34, 39–48.

    Article  CAS  PubMed  Google Scholar 

  33. Madjar, J. J., Michel, S., Cozzone, A. J., and Reboud, J. P. (1979) A method to identify individual proteins in four different two-dimensional electrophoresis systems: application to Escherichia coli ribosomal proteins, Anal. Biochem., 92, 174–182.

    Article  CAS  PubMed  Google Scholar 

  34. Kostareva, O., Tishchenko, S., Nikonova, E., Kljashtorny, V., Nevskaya, N., Nikulin, A., Sycheva, A., Moshkovskii, S., Piendl, W., Garber, M., and Nikonov, S. (2011) Disruption of shape complementarity in the ribosomal protein L1-RNA contact region does not hinder specific recognition of the RNA target site, J. Mol. Recognit., 4, 524–532.

    Article  Google Scholar 

  35. Hess, B., Kutzner, C., van der Spoel, D., and Lindahl, E. (2008) GROMACS 4: Algorithms for highly efficient, loadbalanced, and scalable molecular simulation, J. Chem. Theory Comp., 4, 435–447.

    Article  CAS  Google Scholar 

  36. MacKerell, A. D., Bashford, D., Bellott, M., Dunbrack, R. L., Evanseck, J. D., Field, M. J., Fischer, S., Gao, J., Guo, H., and Ha, S. (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins, J. Phys. Chem. B., 102, 3586–3616.

    CAS  PubMed  Google Scholar 

  37. MacKerell, A. D., Feig, M., and Brooks, C. L. (2004) Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations, J. Comp. Chem., 25, 1400–1415.

    Article  CAS  Google Scholar 

  38. Nevskaya, N. A., Nikonov, O. S., Revtovich, C. V., and Garber, M. B. (2004) Identification of RNA-recognizing modules on the surface of ribosomal proteins, Mol. Biol. (Moscow), 38, 926–936.

    Article  Google Scholar 

  39. Kaczanowska, M., and Ryden-Aulin, M. (2007) Ribosome biogenesis and the translation process in Escherichia coli, Microbiol. Mol. Biol. Rev., 71, 477–494.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Wilson, D. N., and Nierhaus, K. H. (2007) The weird and wonderful world of bacterial ribosome regulation, Crit. Rev. Biochem. Mol. Biol., 42, 187–219.

    CAS  PubMed  Google Scholar 

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Correspondence to G. M. Gongadze.

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Published in Russian in Biokhimiya, 2014, Vol. 79, No. 8, pp. 1031–1041.

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Anikaev, A.Y., Korepanov, A.P., Korobeinikova, A.V. et al. Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo . Biochemistry Moscow 79, 826–835 (2014). https://doi.org/10.1134/S0006297914080112

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  • DOI: https://doi.org/10.1134/S0006297914080112

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