Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1998 Feb 16;17(4):1141–1151. doi: 10.1093/emboj/17.4.1141

Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis.

L Janosi 1, S Mottagui-Tabar 1, L A Isaksson 1, Y Sekine 1, E Ohtsubo 1, S Zhang 1, S Goon 1, S Nelken 1, M Shuda 1, A Kaji 1
PMCID: PMC1170462  PMID: 9463391

Abstract

Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post-termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature-sensitive Escherichia coli mutants which we isolated in this study. At non-permissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re-initiate translation at various sites in all frames without the presence of an initiation codon. Re-initiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo.

Full Text

The Full Text of this article is available as a PDF (348.9 KB).

Selected References

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

  1. Aharonowitz Y., Ron E. Z. A temperature sensitive mutant in Bacillus subtilis with an altered elongation factor G. Mol Gen Genet. 1972;119(2):131–138. doi: 10.1007/BF00269132. [DOI] [PubMed] [Google Scholar]
  2. Björnsson A., Isaksson L. A. UGA codon context which spans three codons. Reversal by ms2i6A37 in tRNA, mutation in rpsD(S4) or streptomycin. J Mol Biol. 1993 Aug 20;232(4):1017–1029. doi: 10.1006/jmbi.1993.1457. [DOI] [PubMed] [Google Scholar]
  3. Björnsson A., Mottagui-Tabar S., Isaksson L. A. Structure of the C-terminal end of the nascent peptide influences translation termination. EMBO J. 1996 Apr 1;15(7):1696–1704. [PMC free article] [PubMed] [Google Scholar]
  4. Blomfield I. C., Vaughn V., Rest R. F., Eisenstein B. I. Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol Microbiol. 1991 Jun;5(6):1447–1457. doi: 10.1111/j.1365-2958.1991.tb00791.x. [DOI] [PubMed] [Google Scholar]
  5. Bult C. J., White O., Olsen G. J., Zhou L., Fleischmann R. D., Sutton G. G., Blake J. A., FitzGerald L. M., Clayton R. A., Gocayne J. D. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science. 1996 Aug 23;273(5278):1058–1073. doi: 10.1126/science.273.5278.1058. [DOI] [PubMed] [Google Scholar]
  6. Cole J. R., Olsson C. L., Hershey J. W., Grunberg-Manago M., Nomura M. Feedback regulation of rRNA synthesis in Escherichia coli. Requirement for initiation factor IF2. J Mol Biol. 1987 Dec 5;198(3):383–392. doi: 10.1016/0022-2836(87)90288-9. [DOI] [PubMed] [Google Scholar]
  7. Cummings H. S., Hershey J. W. Translation initiation factor IF1 is essential for cell viability in Escherichia coli. J Bacteriol. 1994 Jan;176(1):198–205. doi: 10.1128/jb.176.1.198-205.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fowler A. V., Zabin I. The amino acid sequence of beta-galactosidase of Escherichia coli. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1507–1510. doi: 10.1073/pnas.74.4.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fraser C. M., Gocayne J. D., White O., Adams M. D., Clayton R. A., Fleischmann R. D., Bult C. J., Kerlavage A. R., Sutton G., Kelley J. M. The minimal gene complement of Mycoplasma genitalium. Science. 1995 Oct 20;270(5235):397–403. doi: 10.1126/science.270.5235.397. [DOI] [PubMed] [Google Scholar]
  10. Hamilton C. M., Aldea M., Washburn B. K., Babitzke P., Kushner S. R. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol. 1989 Sep;171(9):4617–4622. doi: 10.1128/jb.171.9.4617-4622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hashimoto-Gotoh T., Sekiguchi M. Mutations of temperature sensitivity in R plasmid pSC101. J Bacteriol. 1977 Aug;131(2):405–412. doi: 10.1128/jb.131.2.405-412.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Himmelreich R., Hilbert H., Plagens H., Pirkl E., Li B. C., Herrmann R. Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res. 1996 Nov 15;24(22):4420–4449. doi: 10.1093/nar/24.22.4420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hirashima A., Kaji A. Factor dependent breakdown of polysomes. Biochem Biophys Res Commun. 1970 Nov 25;41(4):877–883. doi: 10.1016/0006-291x(70)90165-8. [DOI] [PubMed] [Google Scholar]
  14. Hirashima A., Kaji A. Factor-dependent release of ribosomes from messenger RNA. Requirement for two heat-stable factors. J Mol Biol. 1972 Mar 14;65(1):43–58. doi: 10.1016/0022-2836(72)90490-1. [DOI] [PubMed] [Google Scholar]
  15. Hirashima A., Kaji A. Purification and properties of ribosome-releasing factor. Biochemistry. 1972 Oct 24;11(22):4037–4044. doi: 10.1021/bi00772a005. [DOI] [PubMed] [Google Scholar]
  16. Hirashima A., Kaji A. Role of elongation factor G and a protein factor on the release of ribosomes from messenger ribonucleic acid. J Biol Chem. 1973 Nov 10;248(21):7580–7587. [PubMed] [Google Scholar]
  17. Hou Y., Lin Y. P., Sharer J. D., March P. E. In vivo selection of conditional-lethal mutations in the gene encoding elongation factor G of Escherichia coli. J Bacteriol. 1994 Jan;176(1):123–129. doi: 10.1128/jb.176.1.123-129.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ichikawa S., Kaji A. Molecular cloning and expression of ribosome releasing factor. J Biol Chem. 1989 Nov 25;264(33):20054–20059. [PubMed] [Google Scholar]
  19. Janosi L., Hara H., Zhang S., Kaji A. Ribosome recycling by ribosome recycling factor (RRF)--an important but overlooked step of protein biosynthesis. Adv Biophys. 1996;32:121–201. doi: 10.1016/0065-227x(96)84743-5. [DOI] [PubMed] [Google Scholar]
  20. Janosi L., Ricker R., Kaji A. Dual functions of ribosome recycling factor in protein biosynthesis: disassembling the termination complex and preventing translational errors. Biochimie. 1996;78(11-12):959–969. doi: 10.1016/s0300-9084(97)86718-1. [DOI] [PubMed] [Google Scholar]
  21. Janosi L., Shimizu I., Kaji A. Ribosome recycling factor (ribosome releasing factor) is essential for bacterial growth. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4249–4253. doi: 10.1073/pnas.91.10.4249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kawakami K., Inada T., Nakamura Y. Conditionally lethal and recessive UGA-suppressor mutations in the prfB gene encoding peptide chain release factor 2 of Escherichia coli. J Bacteriol. 1988 Nov;170(11):5378–5381. doi: 10.1128/jb.170.11.5378-5381.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Keiler K. C., Waller P. R., Sauer R. T. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science. 1996 Feb 16;271(5251):990–993. doi: 10.1126/science.271.5251.990. [DOI] [PubMed] [Google Scholar]
  24. Kozak M. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol Cell Biol. 1987 Oct;7(10):3438–3445. doi: 10.1128/mcb.7.10.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lindahl L., Hinnebusch A. Diversity of mechanisms in the regulation of translation in prokaryotes and lower eukaryotes. Curr Opin Genet Dev. 1992 Oct;2(5):720–726. doi: 10.1016/s0959-437x(05)80132-7. [DOI] [PubMed] [Google Scholar]
  26. Mottagui-Tabar S., Björnsson A., Isaksson L. A. The second to last amino acid in the nascent peptide as a codon context determinant. EMBO J. 1994 Jan 1;13(1):249–257. doi: 10.1002/j.1460-2075.1994.tb06255.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Ogawa K., Kaji A. Requirement for ribosome-releasing factor for the release of ribosomes at the termination codon. Eur J Biochem. 1975 Oct 15;58(2):411–419. doi: 10.1111/j.1432-1033.1975.tb02388.x. [DOI] [PubMed] [Google Scholar]
  29. Olsson C. L., Graffe M., Springer M., Hershey J. W. Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation in Escherichia coli. Mol Gen Genet. 1996 Apr 10;250(6):705–714. doi: 10.1007/BF02172982. [DOI] [PubMed] [Google Scholar]
  30. Pavlov M. Y., Freistroffer D. V., MacDougall J., Buckingham R. H., Ehrenberg M. Fast recycling of Escherichia coli ribosomes requires both ribosome recycling factor (RRF) and release factor RF3. EMBO J. 1997 Jul 1;16(13):4134–4141. doi: 10.1093/emboj/16.13.4134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Richter Dahlfors A. A., Kurland C. G. Novel mutants of elongation factor G. J Mol Biol. 1990 Oct 20;215(4):549–557. doi: 10.1016/s0022-2836(05)80167-6. [DOI] [PubMed] [Google Scholar]
  32. Ryden M., Murphy J., Martin R., Isaksson L., Gallant J. Mapping and complementation studies of the gene for release factor 1. J Bacteriol. 1986 Dec;168(3):1066–1069. doi: 10.1128/jb.168.3.1066-1069.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ryoji M., Berland R., Kaji A. Reinitiation of translation from the triplet next to the amber termination codon in the absence of ribosome-releasing factor. Proc Natl Acad Sci U S A. 1981 Oct;78(10):5973–5977. doi: 10.1073/pnas.78.10.5973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ryoji M., Karpen J. W., Kaji A. Further characterization of ribosome releasing factor and evidence that it prevents ribosomes from reading through a termination codon. J Biol Chem. 1981 Jun 10;256(11):5798–5801. [PubMed] [Google Scholar]
  35. Sekine Y., Eisaki N., Ohtsubo E. Translational control in production of transposase and in transposition of insertion sequence IS3. J Mol Biol. 1994 Feb 4;235(5):1406–1420. doi: 10.1006/jmbi.1994.1097. [DOI] [PubMed] [Google Scholar]
  36. Shimizu I., Kaji A. Identification of the promoter region of the ribosome-releasing factor cistron (frr). J Bacteriol. 1991 Aug;173(16):5181–5187. doi: 10.1128/jb.173.16.5181-5187.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tiedeman A. A., Smith J. M. lacZY gene fusion cassettes with KanR resistance. Nucleic Acids Res. 1988 Apr 25;16(8):3587–3587. doi: 10.1093/nar/16.8.3587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Vizcaíno N., Cloeckaert A., Dubray G., Zygmunt M. S. Cloning, nucleotide sequence, and expression of the gene coding for a ribosome releasing factor-homologous protein of Brucella melitensis. Infect Immun. 1996 Nov;64(11):4834–4837. doi: 10.1128/iai.64.11.4834-4837.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wada A., Igarashi K., Yoshimura S., Aimoto S., Ishihama A. Ribosome modulation factor: stationary growth phase-specific inhibitor of ribosome functions from Escherichia coli. Biochem Biophys Res Commun. 1995 Sep 14;214(2):410–417. doi: 10.1006/bbrc.1995.2302. [DOI] [PubMed] [Google Scholar]
  40. Yamagishi M., Matsushima H., Wada A., Sakagami M., Fujita N., Ishihama A. Regulation of the Escherichia coli rmf gene encoding the ribosome modulation factor: growth phase- and growth rate-dependent control. EMBO J. 1993 Feb;12(2):625–630. doi: 10.1002/j.1460-2075.1993.tb05695.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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

RESOURCES