Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Functional Analysis of the Residue 789 in Escherichia coli 16S rRNA and Development of a Method to Select Second-site Revertants

Escherichia coli 16S rRNA의 789 염기의 기능분석 및 이차복귀돌연변이체 발췌를 위한 방법 개발

  • Kim Jong-Myung (Department of Life Science, Chung-Ang University) ;
  • Go Ha-Young (Department of Life Science, Chung-Ang University) ;
  • Song Woo-Seok (Department of Life Science, Chung-Ang University) ;
  • Ryou Sang-Mi (Department of Life Science, Chung-Ang University) ;
  • Lee Kang-Seok (Department of Life Science, Chung-Ang University)
  • 김종명 (중앙대학교 자연과학대학 생명과학과) ;
  • 고하영 (중앙대학교 자연과학대학 생명과학과) ;
  • 송우석 (중앙대학교 자연과학대학 생명과학과) ;
  • 류상미 (중앙대학교 자연과학대학 생명과학과) ;
  • 이강석 (중앙대학교 자연과학대학 생명과학과)
  • Published : 2006.06.01
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Abstract

A base substitution was introduced at the position 789 in Escherichia coli 16S rRNA, which was previously identified as an invariant residue for ribosome function and the ability of the mutant ribosomes to translate chloramphenicol acetyltransfernse mRNA was measured by determining the degree of resistance to chloramphenicol of cells expressing these mutant ribosomes. As expected, mutant ribosomes containing a base sub-stitution at the position 789 showed significantly reduced protein-synthesis ability and to identify a functional role played by this residue in protein synthesis, we developed an efficient genetic method to select second-site revertants in 16S rRNA that restore protein-synthesis function to these mutant ribosomes.

Escherichia coli 16S rRNA의 잘 보존된 부분인 790 loop의 즉흥진화를 통한 분석에서 리보솜의 단백질 수행기능을 위해서 필수불가결한 것으로 추측되는 789번 위치에 염기치환을 유발하여 제작한 변이체 리보솜의 기능을 chloramphenicol acetyltransfernse mRNA의 단백질로의 번역능력 차이에 따른 chloramphenicol에 대한 저항성의 정도를 측정함으로써 분석하였다. 예상했던 바와 같이 모든 변이체 리보솜의 단백질 합성능력은 현저히 저하되었으며, 789 염기의 단백질합성에서의 기능을 규명하기 위하여 16S rRNA 변이체의 기능을 회복시키는 이차복귀돌연변이(second-site revertant)를 발췌하는 효과적인 유전학적 실험방법을 개발하였다.

Keywords

References

  1. Cannone, J. J., S. Subramanian, M.N. Schnare, J. R. Collett, L.M. D'Souza, Y. Du, B. Feng, N. Lin, L.V. Madabusi, K.M. Muller, N. Pande, Z. Shang, N. Yu, and R.R. Gutell. 2002. The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs. BMC Bioinformatics 3, 2 https://doi.org/10.1186/1471-2105-3-2
  2. Cate, J.H., M.M. Yusupov, G. Z. Yusupova, T.N. Earnest, and H. F. Noller. 1999. X-ray crystal structures of 70S ribosome functional complexes. Science 285, 2095-2104 https://doi.org/10.1126/science.285.5436.2095
  3. Cech, T.R., A.J. Zaug, and P.J. Grabowski. 1981. In vitro splicing of the ribosomal RNA precursor of Tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence. Cell 27, 487-96 https://doi.org/10.1016/0092-8674(81)90390-1
  4. Clemons, W.M. Jr., J.L. May, B.T. Wimberly, J.P. McCutcheon, M. S. Capel, and V. Ramakrishnan. 1999. Structure of a bacterial 30S ribosomal subunit at 5.5 A resolution. Nature 400, 833-40 https://doi.org/10.1038/23631
  5. Dahlberg, A.E. 1989. The functional role of ribosomal RNA in protein synthesis. Cell 57, 525-529 https://doi.org/10.1016/0092-8674(89)90122-0
  6. Dinos, G., D.N. Wilson, Y. Teraoka, W. Szaflarski, P. Fucini, D. Kalpaxis, and K.H. Nierhaus. 2004. Dissecting the ribosomal inhibition mechanisms of edeine and pactamycin: the universally conserved residues G693 and C795 regulate P-site RNA binding. Mol. Cell. 13, 113-24 https://doi.org/10.1016/S1097-2765(04)00002-4
  7. Gram, H., L.A. Marconi, C.F. Barbas 3rd, T.A. Collet, R.A. Lerner, and A. S. Kang. 1992. In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library. Proc. Natl. Acad. Sci. USA 89, 3576-80
  8. Gutell, R.R., N. Larsen, and C.R. Woese. 1994. Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective. Microbiol. Rev. 58, 10-26
  9. Higuchi, R. 1989. Using PCR to engineer DNA. p. 61-70. In PCR Technology (Erlich, H.A., ed.), Stockton Press, New York
  10. Lee, K., C.A. Holland-Staley, and P.R. Cunningham. 1996. Genetic analysis of the Shine-Dalgarno interaction: selection of alternative functional mRNA-rRNA combinations. RNA 2, 1270- 1285
  11. 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
  12. 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
  13. Moazed, D. and H.F. Noller. 1986. Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes. Cell 47, 985-994 https://doi.org/10.1016/0092-8674(86)90813-5
  14. Moazed, D. and H.F. Noller. 1987. Interaction of antibiotics with functional sites in 16S ribosomal RNA. Nature 327, 389-394 https://doi.org/10.1038/327389a0
  15. Moazed, D., R.R. Samaha, C. Gualerzi, and H.F. Noller. 1995. Specific protection of 16 S rRNA by translational initiation factors. J. Mol. Biol. 248, 207-210
  16. Noller, H.F. 1991. Ribosomal RNA and translation. Ann. Rev. Biochem. 60, 191-227 https://doi.org/10.1146/annurev.bi.60.070191.001203
  17. Szatkiewicz, J. P., H. Cho, S.-M. Ryou, J.-M. Kim, P. R. Cunningham, and K. Lee. 2006. Genetic analysis of a structural motif within the conserved 530 stem-loop of Escherichia coli 16S rRNA. J. Mocrobiol. Biotechnol. 16, 569-575
  18. Tapprich, W.E., D. J. Goss, and A.E. Dahlberg. 1989. Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis. Proc. Natl. Acad. Sci. USA 86, 4927-4931