Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1998 Mar 2;17(5):1507–1514. doi: 10.1093/emboj/17.5.1507

Mutations in RNAs of both ribosomal subunits cause defects in translation termination.

A L Arkov 1, D V Freistroffer 1, M Ehrenberg 1, E J Murgola 1
PMCID: PMC1170498  PMID: 9482747

Abstract

Mutations in RNAs of both subunits of the Escherichia coli ribosome caused defects in catalysis of peptidyl-tRNA hydrolysis in a realistic in vitro termination system. Assaying the two codon-dependent cytoplasmic proteins that drive termination, RF1 and RF2, we observed large defects with RF2 but not with RF1, a result consistent with the in vivo properties of the mutants. Our study presents the first direct in vitro evidence demonstrating the involvement of RNAs from both the large and the small ribosomal subunits in catalysis of peptidyl-tRNA hydrolysis during termination of protein biosynthesis. The results and conclusions are of general significance since the rRNA nucleotides studied have been virtually universally conserved throughout evolution. Our findings suggest a novel role for rRNAs of both subunits as molecular transmitters of a signal for termination.

Full Text

The Full Text of this article is available as a PDF (338.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brot N., Tate W. P., Caskey C. T., Weissbach H. The requirement for ribosomal proteins L7 and L12 in peptide-chain termination. Proc Natl Acad Sci U S A. 1974 Jan;71(1):89–92. doi: 10.1073/pnas.71.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brown C. M., McCaughan K. K., Tate W. P. Two regions of the Escherichia coli 16S ribosomal RNA are important for decoding stop signals in polypeptide chain termination. Nucleic Acids Res. 1993 May 11;21(9):2109–2115. doi: 10.1093/nar/21.9.2109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Caskey C. T., Beaudet A. L., Scolnick E. M., Rosman M. Hydrolysis of fMet-tRNA by peptidyl transferase. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3163–3167. doi: 10.1073/pnas.68.12.3163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caskey C. T., Bosch L., Konecki D. S. Release factor binding to ribosome requires an intact 16 S rRNA 3' terminus. J Biol Chem. 1977 Jul 10;252(13):4435–4437. [PubMed] [Google Scholar]
  5. Caskey C. T., Tompkins R., Scolnick E., Caryk T., Nirenberg M. Sequential translation of trinucleotide codons for the initiation and termination of protein synthesis. Science. 1968 Oct 4;162(3849):135–138. doi: 10.1126/science.162.3849.135. [DOI] [PubMed] [Google Scholar]
  6. Chernoff Y. O., Newnam G. P., Liebman S. W. The translational function of nucleotide C1054 in the small subunit rRNA is conserved throughout evolution: genetic evidence in yeast. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2517–2522. doi: 10.1073/pnas.93.6.2517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dahlberg A. E. The functional role of ribosomal RNA in protein synthesis. Cell. 1989 May 19;57(4):525–529. doi: 10.1016/0092-8674(89)90122-0. [DOI] [PubMed] [Google Scholar]
  8. Freistroffer D. V., Pavlov M. Y., MacDougall J., Buckingham R. H., Ehrenberg M. Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP-dependent manner. EMBO J. 1997 Jul 1;16(13):4126–4133. doi: 10.1093/emboj/16.13.4126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Godson G. N., Sinsheimer R. L. Use of Brij lysis as a general method to prepare polyribosomes from Escherichia coli. Biochim Biophys Acta. 1967 Dec 19;149(2):489–495. doi: 10.1016/0005-2787(67)90176-1. [DOI] [PubMed] [Google Scholar]
  10. Goldstein J. L., Caskey C. T. Peptide chain termination: effect of protein S on ribosomal binding of release factors. Proc Natl Acad Sci U S A. 1970 Oct;67(2):537–543. doi: 10.1073/pnas.67.2.537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gourse R. L., Takebe Y., Sharrock R. A., Nomura M. Feedback regulation of rRNA and tRNA synthesis and accumulation of free ribosomes after conditional expression of rRNA genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1069–1073. doi: 10.1073/pnas.82.4.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jelenc P. C., Kurland C. G. Nucleoside triphosphate regeneration decreases the frequency of translation errors. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3174–3178. doi: 10.1073/pnas.76.7.3174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jelenc P. C. Rapid purification of highly active ribosomes from Escherichia coli. Anal Biochem. 1980 Jul 1;105(2):369–374. doi: 10.1016/0003-2697(80)90472-8. [DOI] [PubMed] [Google Scholar]
  14. Jemiolo D. K., Pagel F. T., Murgola E. J. UGA suppression by a mutant RNA of the large ribosomal subunit. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12309–12313. doi: 10.1073/pnas.92.26.12309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maidak B. L., Olsen G. J., Larsen N., Overbeek R., McCaughey M. J., Woese C. R. The Ribosomal Database Project (RDP). Nucleic Acids Res. 1996 Jan 1;24(1):82–85. doi: 10.1093/nar/24.1.82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mikuni O., Kawakami K., Nakamura Y. Sequence and functional analysis of mutations in the gene encoding peptide-chain-release factor 2 of Escherichia coli. Biochimie. 1991 Dec;73(12):1509–1516. doi: 10.1016/0300-9084(91)90185-4. [DOI] [PubMed] [Google Scholar]
  17. Moazed D., Noller H. F. Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites. Cell. 1989 May 19;57(4):585–597. doi: 10.1016/0092-8674(89)90128-1. [DOI] [PubMed] [Google Scholar]
  18. Moine H., Dahlberg A. E. Mutations in helix 34 of Escherichia coli 16 S ribosomal RNA have multiple effects on ribosome function and synthesis. J Mol Biol. 1994 Oct 28;243(3):402–412. doi: 10.1006/jmbi.1994.1668. [DOI] [PubMed] [Google Scholar]
  19. Murgola E. J., Hijazi K. A., Göringer H. U., Dahlberg A. E. Mutant 16S ribosomal RNA: a codon-specific translational suppressor. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4162–4165. doi: 10.1073/pnas.85.12.4162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Murgola E. J., Pagel F. T., Hijazi K. A., Arkov A. L., Xu W., Zhao S. Q. Variety of nonsense suppressor phenotypes associated with mutational changes at conserved sites in Escherichia coli ribosomal RNA. Biochem Cell Biol. 1995 Nov-Dec;73(11-12):925–931. doi: 10.1139/o95-100. [DOI] [PubMed] [Google Scholar]
  21. Nakamura Y., Ito K., Isaksson L. A. Emerging understanding of translation termination. Cell. 1996 Oct 18;87(2):147–150. doi: 10.1016/s0092-8674(00)81331-8. [DOI] [PubMed] [Google Scholar]
  22. Noller H. F., Hoffarth V., Zimniak L. Unusual resistance of peptidyl transferase to protein extraction procedures. Science. 1992 Jun 5;256(5062):1416–1419. doi: 10.1126/science.1604315. [DOI] [PubMed] [Google Scholar]
  23. Noller H. F. Ribosomal RNA and translation. Annu Rev Biochem. 1991;60:191–227. doi: 10.1146/annurev.bi.60.070191.001203. [DOI] [PubMed] [Google Scholar]
  24. Noller H. F., Woese C. R. Secondary structure of 16S ribosomal RNA. Science. 1981 Apr 24;212(4493):403–411. doi: 10.1126/science.6163215. [DOI] [PubMed] [Google Scholar]
  25. Pagel F. T., Zhao S. Q., Hijazi K. A., Murgola E. J. Phenotypic heterogeneity of mutational changes at a conserved nucleotide in 16 S ribosomal RNA. J Mol Biol. 1997 Apr 18;267(5):1113–1123. doi: 10.1006/jmbi.1997.0943. [DOI] [PubMed] [Google Scholar]
  26. Rosendahl G., Douthwaite S. The antibiotics micrococcin and thiostrepton interact directly with 23S rRNA nucleotides 1067A and 1095A. Nucleic Acids Res. 1994 Feb 11;22(3):357–363. doi: 10.1093/nar/22.3.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sigmund C. D., Ettayebi M., Borden A., Morgan E. A. Antibiotic resistance mutations in ribosomal RNA genes of Escherichia coli. Methods Enzymol. 1988;164:673–690. doi: 10.1016/s0076-6879(88)64077-8. [DOI] [PubMed] [Google Scholar]
  28. Tate W. P., Brown C. M. Translational termination: "stop" for protein synthesis or "pause" for regulation of gene expression. Biochemistry. 1992 Mar 10;31(9):2443–2450. doi: 10.1021/bi00124a001. [DOI] [PubMed] [Google Scholar]
  29. Tate W. P., Dalphin M. E., Pel H. J., Mannering S. A. The stop signal controls the efficiency of release factor-mediated translational termination. Genet Eng (N Y) 1996;18:157–182. doi: 10.1007/978-1-4899-1766-9_10. [DOI] [PubMed] [Google Scholar]
  30. Tate W. P., Mannering S. A. Three, four or more: the translational stop signal at length. Mol Microbiol. 1996 Jul;21(2):213–219. doi: 10.1046/j.1365-2958.1996.6391352.x. [DOI] [PubMed] [Google Scholar]
  31. Tate W. P., Poole E. S., Mannering S. A. Hidden infidelities of the translational stop signal. Prog Nucleic Acid Res Mol Biol. 1996;52:293–335. doi: 10.1016/s0079-6603(08)60970-8. [DOI] [PubMed] [Google Scholar]
  32. Thompson J., Cundliffe E. The binding of thiostrepton to 23S ribosomal RNA. Biochimie. 1991 Jul-Aug;73(7-8):1131–1135. doi: 10.1016/0300-9084(91)90156-u. [DOI] [PubMed] [Google Scholar]
  33. Van de Peer Y., Chapelle S., De Wachter R. A quantitative map of nucleotide substitution rates in bacterial rRNA. Nucleic Acids Res. 1996 Sep 1;24(17):3381–3391. doi: 10.1093/nar/24.17.3381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vogel Z., Zamir A., Elson D. The possible involvement of peptidyl transferase in the termination step of protein biosynthesis. Biochemistry. 1969 Dec;8(12):5161–5168. doi: 10.1021/bi00840a070. [DOI] [PubMed] [Google Scholar]
  35. Xu W., Murgola E. J. Functional effects of mutating the closing GxA base-pair of a conserved hairpin loop in 23 S ribosomal RNA. J Mol Biol. 1996 Dec 6;264(3):407–411. doi: 10.1006/jmbi.1996.0649. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES