Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Development of Genetic System for Isolation of SSU rRNA Mutants that Bypass SecM-Mediated Ribosome Stalling

SecM에서 유래한 접착펩타이드에 의한 라이보솜 정지를 우회하는 SSU rRNA 돌연변이체 발굴을 위한 유전학적 시스템 개발

  • 하혜정 (중앙대학교 자연과학대학 생명과학과) ;
  • 김홍만 (중앙대학교 자연과학대학 생명과학과) ;
  • 염지현 (중앙대학교 자연과학대학 생명과학과) ;
  • 이강석 (중앙대학교 자연과학대학 생명과학과)
  • Published : 2008.12.31
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Abstract

Ribosome stalling by nascent sticky peptide has been reported in several organisms across the kingdom. To test whether small subunit (SSU) rRNA is involved in this phenomenon, we developed a genetic system that utilized the specialized ribosome system to isolate SSU rRNA mutants that enable ribosomes to bypass the SecM-derived sticky peptide in protein synthesis. In this system, CAT-SecM mRNA, which encodes CAT protein containing the sticky peptide derived from SecM, is only translated by specialized ribosomes. These ribosomes were shown to transiently stall on CAT-SecM mRNA followed by the synthesis of the sticky peptide. Expression of specialized ribosomes resulted in the decreased steady-state level of CAT-SecM mRNA, which is consistent with a notion that ribosome stalling induces mRNA degradation. Isolation and characterization of SSU rRNA mutations using this genetic system that are sufficient to circumvent ribosome stalling induced by the SecM-derived sticky peptide will provide evidence of SSU rRNA function in mRNA cleavage.

최근 단백질 합성 과정 중 라이보솜의 일시적인 정지에 의한 라이보솜의 A자리에서 전사체가 분해되는 현상이 여러 생명체에서 보고되었다. 이러한 현상이 라이보솜의 작은 소단위체를 이루고 있는 SSU rRNA의 기능과 관련 있는지를 알아보기 위해, SecM에서 유래한 접착펩타이드에 의한 라이보솜 정지를 우회하는 SSU rRNA 돌연변이체 발굴을 위한 유전학적 시스템을 개발하였다. 이 시스템에서는 SecM에서 유래한 접착펩타이를 포함하는 CAT 단백질을 코딩하는 CAT-SecM 전사체가 플라스미드에서 유래한 SSU rRNA를 포함한 재조합 라이보솜에 의해서만 해독된다. 이러한 재조합 라이보솜은 접착펩타이드를 합성한 후 CAT-SecM mRNA 상에서 일시 정체하며, 재조합 라이보솜의 발현은 이 전사체의 양을 감소시키는 것을 확인하였다. 이러한 결과는 개발된 시스템을 이용해 라이보솜 검지를 우회하는 SSU rRNA 돌연변이체의 선별이 가능하다는 것을 보여주며, 이러한 변이체에 대한 연구는 단백질 합성 단계에서 일어나는 라이보솜 정지와 전사체 절단 현상에 있어서, SSU rRNA의 역할을 규명하는데 기여할 것이다.

Keywords

References

  1. Brosius, J., T. Dull, D. Sleeter, and H.F. Noller. 1981. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J. Mol. Biol. 148, 107-127 https://doi.org/10.1016/0022-2836(81)90508-8
  2. Calos, M.P. 1978. DNA sequence for a low-level promoter of the lac repressor gene and an 'up' promoter mutation. Nature 274, 762-765 https://doi.org/10.1038/274762a0
  3. Carrier, T.A. and J.D. Keasling. 1997. Mechanistic modeling of prokaryotic mRNA decay. J. Theor. Biol. 189, 195-209 https://doi.org/10.1006/jtbi.1997.0509
  4. Deana, A. and J.G. Belasco. 2005. Lost in translation: the influence of ribosomes on bacterial mRNA decay. Genes Dev. 19, 2526-2533 https://doi.org/10.1101/gad.1348805
  5. Doma, M.K. and R. Parker. 2006. Endonucleolytic cleavage of eukaryotic mRNAs with stalls in translation elongation. Nature 440, 561-564 https://doi.org/10.1038/nature04530
  6. Drider, D., J.M. DiChiara, J. Wei, J.S. Sharp, and D.H. Bechhofer. 2002. Endonuclease cleavage of messenger RNA in Bacillus subtilis. Mol. Microbiol. 43, 1319-1329 https://doi.org/10.1046/j.1365-2958.2002.02830.x
  7. Gaba, A., Z. Wang, T. Krishnamoorthy, A.G. Hinnebusch, and M.S. Sachs. 2001. Physical evidence for distinct mechanisms of translational control by upstream open reading frames. EMBO J. 20, 6453-6463 https://doi.org/10.1093/emboj/20.22.6453
  8. Gatfield, D. and E. Izaurralde. 2004. Nonsense-mediated messenger RNA decay is initiated by endonucleolytic cleavage in Drosophila. Nature 429, 575-578 https://doi.org/10.1038/nature02559
  9. Geballe, A.P. and M.S. Sachs. 2000. Translational control by upstream open reading frames, p. 595-614. In N. Sonenberg, J.W.B. Hershey, and M.B. Mathews (eds.), Translational control of gene epression. Cold Spring Harbor Laboratory Press, New York, N.Y., USA
  10. Hartz, D., D.S. McPheeters, R. Traut, and L. Gold. 1988. Extension inhibition analysis of translation initiation complexes. Methods Enzymol. 164, 419-425 https://doi.org/10.1016/S0076-6879(88)64058-4
  11. Hayes, C.S. and R.T. Sauer. 2003. Cleavage of the A site mRNA codon during ribosome pausing provides a mechanism for translation quality control. Mol. Cell. 12, 903-911 https://doi.org/10.1016/S1097-2765(03)00385-X
  12. Karzai, A.W., E.D. Roche, and R.T. Sauer. 2000. The SsrA-SmpB system for protein tagging, directed degradation and ribosome rescue. Nat. Struct. Biol. 7, 449-455 https://doi.org/10.1038/75843
  13. Keiler, K.C., P.R. Waller, and R.T. Sauer. 1996. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271, 990-993 https://doi.org/10.1126/science.271.5251.990
  14. Lee, K., C.A. Holland-Staley, and P.R. Cunningham. 2001. Genetic approaches to studying protein synthesis: Effects of mutations at $\Psi$516 and A535 in Escherichia coli 16S rRNA J. Nutr. 131, 2994S-3004S https://doi.org/10.1093/jn/131.11.2994S
  15. Lee, K., C.A. Holland-Stanley, and P.R. Cunningham. 1996. Genetic analysis of the Shine-Dalgarno interaction: selection of alternative functional mRNA-rRNA combinations. RNA 2, 1270-1285
  16. Lee, K., S. Varma, J. SantaLucia, and P. Cunningham. 1997. In vivo determination of DNA structure-function relationships: analysis of the 790 loop in ribosomal RNA. J. Mol. Biol. 269, 732-743 https://doi.org/10.1006/jmbi.1997.1092
  17. Lee, N., C. Francklyn, and E.P. Hamilton. 1987. Arabinoseinduced binding of AraC protein to araI2 activates the araBAD operon promoter. PNAS 88, 8814-8818
  18. Lopez, P.J., I. Marchand, O. Yarchuk, and M. Dreyfus. 1998. Translation inhibitors stabilize Escherichia coli mRNAs independently of ribosome protection. PNAS 95, 6067-6072
  19. Lovett, P.S. and E.J. Rogers. 1996. Ribosome regulation by the nascent peptide. Microbiol. Rev. 60, 366-385
  20. Mori, H. and K. Ito. 2001. The Sec protein-translocation pathway. Trends Microbiol. 9, 494-500 https://doi.org/10.1016/S0966-842X(01)02174-6
  21. Morris, D.R. and A.P. Geballe. 2000. Upstream open reading frames as regulators of mRNA translation. Mol. Cell. Biol. 20, 8635-8642 https://doi.org/10.1128/MCB.20.23.8635-8642.2000
  22. Murakami, A., H. Nakatogawa, and K. Ito. 2004. Translation arrest of SecM is essential for the basal and regulated expression of SecA. PNAS 101, 12330-12335
  23. Nakatogawa, H. and K. Ito. 2001. Secretion monitor, SecM, undergoes self-translation arrest in the cytosol. Mol. Cell. 7, 185-192 https://doi.org/10.1016/S1097-2765(01)00166-6
  24. Nakatogawa, H. and K. Ito. 2002. The ribosomal exit tunnel functions as a discriminating gate. Cell 108, 629-636 https://doi.org/10.1016/S0092-8674(02)00649-9
  25. Oliver, D.B. and J. Beckwith. 1982. Regulation of a membrane component required for protein secretion in Escherichia coli. Cell 30, 311-319 https://doi.org/10.1016/0092-8674(82)90037-X
  26. Onouchi, H., Y. Nagami, Y. Haraguchi, M. Nakamoto, Y. Nishimura, R. Sakurai, N. Nagao, D. Kawasaki, Y. Kadokura, and S. Naito. 2005. Nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in the CGS1 gene of Arabidopsis. Genes Dev. 19, 1799-1810 https://doi.org/10.1101/gad.1317105
  27. Powers, T. and H.F. Noller. 1991. A functional pseudoknot in 16S ribosomal RNA. EMBO J. 10, 2203-2214
  28. Raney, A., G.L. Law, G.J. Mize, and D.R. Morris. 2002. Regulated translation termination at the upstream open reading frame in sadenosylmethionine decarboxylase mRNA. J. Biol. Chem. 277, 5988-5994 https://doi.org/10.1074/jbc.M108375200
  29. Sachs, M.S. and A.P. Geballe. 2002. Biochemistry. Sense and sensitivity- controlling the ribosome. Science 297, 1820-1821 https://doi.org/10.1126/science.1076865
  30. Sunohara, T., K. Jojima, H. Tagami, T. Inada, and H. Aiba. 2004. Ribosome stalling during translation elongation induced cleavage of mRNA being translated in Escherichia coli. J. Biol. Chem. 279, 15368-15375 https://doi.org/10.1074/jbc.M312805200
  31. Tenson, T. and M. Ehrenberg. 2002. Regulatory nascent peptides in the ribosomal tunnel. Cell 108, 591-594 https://doi.org/10.1016/S0092-8674(02)00669-4
  32. Vilela, C. and J.E. McCarthy. 2003. Regulation of fungal gene expression via short open reading frames in the mRNA 5' untranslated region. Mol. Microbiol. 49, 859-867 https://doi.org/10.1046/j.1365-2958.2003.03622.x
  33. Withey, J.H. and D.I. Friedman. 2002. The biological roles of trans-translation. Curr. Opin. Microbiol. 5, 154-159 https://doi.org/10.1016/S1369-5274(02)00299-0
  34. Yeom, J.H. and K. Lee. 2006. RraA rescues Escherichia coli cells over-producing RNase E from growth arrest by modulating the ribonucleolytic activity. Biochem. Biophys. Res. Commun. 345, 1372-1376 https://doi.org/10.1016/j.bbrc.2006.05.018