Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Functional Analysis and Selection of Second-site Revertant of Escherichia coli 16S rRNA of C770G

Escherichia coli 16S rRNA 상의 770 위치에 염기치환을 가진 변이체 리보솜의 단백질 합성 능력을 회복시키는 이차복귀돌연변이체의 발췌

  • 하혜정 (중앙대학교 자연과학대학 생명과학과) ;
  • 류상미 (중앙대학교 자연과학대학 생명과학과) ;
  • 이강석 (중앙대학교 자연과학대학 생명과학과) ;
  • 전체옥 (중앙대학교 자연과학대학 생명과학과)
  • Received : 2010.10.18
  • Accepted : 2011.02.20
  • Published : 2011.03.28
Download PDF
⟨ Previous Next ⟩

Abstract

It has been shown that a nucleotide substitution at position 770 in Escherichia coli 16S rRNA, which is implicated in forming the evolutionary conserved B2c intersubunit bridge, has a detrimental effect on ribosome function. In order to isolate second-site revertants that complement ribosomes containing C770G, we performed a random mutagenesis of the 16S rRNA gene and selected clones that could produce more CAT protein translated by specialized ribosome. One of the clones contained two nucleotide substitutions at positions 569 and 904 (C569G and U904C) and these mutations partially complemented the loss of protein-synthesis ability caused by C770G. Further studies using the isolated revertant will provide information about which part of 16S rRNA is interacting with C770 and the consequence of the structure formed by these interactions in the process of protein synthesis.

대장균의 16S rRNA 염기 중 진화적으로 매우 보존되어 있는 B2c 인터브리지의 구성요소 중 하나인 C770염기에 치환을 일으키면 단백질 합성이 저하되는 것으로 알려져 있다. 이 연구에서는 770 위치에 C에서 G로 염기치환(C770G)된 16S rRNA의 기능을 회복시키는 이차복귀돌연변이(secondsite revertant)를 얻기 위해 16S rRNA를 암호화하는 DNA 부분에 무작위로 염기치환을 유발시켜, 재조합 리보솜이 번역하는 CAT mRNA로부터의 단백질 합성능력이 향상된 클론을 선별하였다. 이 실험으로 C770G 염기치환을 가진 변이체 리보솜의 단백질 합성능력을 일부 회복시키는 하나의 이차복귀돌연변이체를 획득하였으며, DNA 염기분석을 통하여 C569G와 U904C 염기치환을 가진 것을 확인하였다. 이러한 연구결과를 이용하여 770 염기가 단백질 합성 과정에서 16S rRNA의 어떤 다른 부분과 결합을 하는지, 또한 그러한 결합으로 이루어지는 구조가 가지게 되는 기능은 무엇인지 등에 대한 리보솜의 구체적인 단백질 합성기작 연구에 도움이 될 것으로 기대한다.

Keywords

References

  1. 김종명, 고하영, 송우석, 류상미, 이강석. 2006. Escherichia coli 16S rRNA의 789 염기의 기능분석 및 이차복귀돌연 변이체 발췌를 위한 방법 개발. 미생물학회지. 42: 156-159.
  2. Ban, N., P. Nissen, J. Hansen, P. B. Moore, and T. A. Steitz. 2000. The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science 289: 905-920. https://doi.org/10.1126/science.289.5481.905
  3. 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
  4. 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
  5. Chapman, N. M. and H. F. Noller. 1977. Protection of specific sites in 16S RNA from chemical modification by association of 30S and 50S ribosomes. J. Mol. Biol. 109: 131-149. https://doi.org/10.1016/S0022-2836(77)80049-1
  6. Gabashvili, I. S., R. K. Agrawal, C. M. Spahn, R. A. Grassucci, D. I. Svergun, J. Frank, and P. Penczek. 2000. Solution structure of the E. coli 70S ribosome at 11.5 A resolution. Cell 100: 537-549. https://doi.org/10.1016/S0092-8674(00)80690-X
  7. Gao, H., J. Sengupta, and M. Valle et al. 2003. Study of the structural dynamics of the E. coli 70S ribosome using realspace refinement. Cell 113: 789-801. https://doi.org/10.1016/S0092-8674(03)00427-6
  8. Hennelly, S. P., A. Antoun, M. Ehrenberg, C. O. Gualerzi, W. Knight, J. S. Lodmell, and W. E. Hill. 2005. A timeresolved investigation of ribosomal subunit association. J. Mol. Biol. 346: 1243-1258. https://doi.org/10.1016/j.jmb.2004.12.054
  9. Herr, W. and H. F. Noller. 1979. Protection of specific sites in 23S and 5S RNA from chemical modification by association of 30S and 50S ribosomes. J. Mol. Biol. 130: 421-432. https://doi.org/10.1016/0022-2836(79)90432-7
  10. Kim, H.-M., J.-H. Yeom, H.-J. Ha, J.-M. Kim, and K. Lee. 2007. Functional analysis of the residues C770 and G771 of E. coli 16S rRNA implicated in forming the intersubunit bridge B2c of the ribosome. J. Microbiol. Biotechnol. 17(7): 1204-1207.
  11. Lee, K., C. A. Holland-Staley, and P. R. Cunningham. 2001. Genetic approaches to studying protein system: effects of mutations at W516 and A 535 in Escherichia coli 16S rRNA. J. Nutr. 131: 2994S-3004S. https://doi.org/10.1093/jn/131.11.2994S
  12. Lee, K., S. Varma, J. Santalucia Jr., and P. R. Cunningham. 1997. In vivo determination of RNA 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
  13. Merryman, C., D. Moazed, G. Daubresse, and H. F. Noller. 1999. Nucleotides in 23S rRNA protected by the association of 30S and 50S ribosomal subunits. J. Mol. Biol. 285: 107-113. https://doi.org/10.1006/jmbi.1998.2243
  14. Schluenzen, F., A. Tocilj, and R. Zarivach et al. 2000. Structure of functionally activated small ribosomal subunit at 3.3 angstroms resolution. Cell 102: 615-623. https://doi.org/10.1016/S0092-8674(00)00084-2
  15. Schuwirth, B. S., M. A. Borovinskaya, C. W. Hau, W. Zhang, A. Vila-Sanjurjo, J. M. Holton, and J. H. Cate. 2005. Structures of the bacterial ribosome at 3.5 A resolution. Science 310: 827-834. https://doi.org/10.1126/science.1117230
  16. Selmer, M., C. M. Dunham, F. V. Murphy 4th, A. Weixlbaumer, S. Petry, A. C. Kelley, J. R. Weir, and V. Ramakrishnan. 2006. Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313: 1935-1942. https://doi.org/10.1126/science.1131127
  17. Spahn, C. M., E. Jan, A. Mulder, R. A. Grassucci, P. Sarnow, and J. Frank. 2004. Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes: The IRES functions as an RNA-based translation factor. Cell 118: 465-475. https://doi.org/10.1016/j.cell.2004.08.001
  18. Wilson, D. N., F. Schluenzen, J. M. Harms, T. Yoshida, T. Ohkubo, R. Albrecht, J. Buerger, Y. Kobayashi, and P. Fucini. 2005. X-ray crystallography study on ribosome recycling: The mechanism of binding and action of RRF on the 50S ribosomal subunit. EMBO J. 24: 251-260. https://doi.org/10.1038/sj.emboj.7600525
  19. 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