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. 1998 Feb 2;17(3):808–816. doi: 10.1093/emboj/17.3.808

Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome.

V Heurgué-Hamard 1, R Karimi 1, L Mora 1, J MacDougall 1, C Leboeuf 1, G Grentzmann 1, M Ehrenberg 1, R H Buckingham 1
PMCID: PMC1170429  PMID: 9451005

Abstract

Peptidyl-tRNA dissociation from ribosomes is an energetically costly but apparently inevitable process that accompanies normal protein synthesis. The drop-off products of these events are hydrolysed by peptidyl-tRNA hydrolase. Mutant selections have been made to identify genes involved in the drop-off of peptidyl-tRNA, using a thermosensitive peptidyl-tRNA hydrolase mutant in Escherichia coli. Transposon insertions upstream of the frr gene, which encodes RF4 (ribosome release or recycling factor), restored growth to this mutant. The insertions impaired expression of the frr gene. Mutations inactivating prfC, encoding RF3 (release factor 3), displayed a similar phenotype. Conversely, production of RF4 from a plasmid increased the thermosensitivity of the peptidyl-tRNA hydrolase mutant. In vitro measurements of peptidyl-tRNA release from ribosomes paused at stop signals or sense codons confirmed that RF3 and RF4 were able to stimulate peptidyl-tRNA release from ribosomes, and showed that this action of RF4 required the presence of translocation factor EF2, known to be needed for the function of RF4 in ribosome recycling. When present together, the three factors were able to stimulate release up to 12-fold. It is suggested that RF4 may displace peptidyl-tRNA from the ribosome in a manner related to its proposed function in removing deacylated tRNA during ribosome recycling.

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Selected References

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

  1. Agris P. F., Guenther R., Ingram P. C., Basti M. M., Stuart J. W., Sochacka E., Malkiewicz A. Unconventional structure of tRNA(Lys)SUU anticodon explains tRNA's role in bacterial and mammalian ribosomal frameshifting and primer selection by HIV-1. RNA. 1997 Apr;3(4):420–428. [PMC free article] [PubMed] [Google Scholar]
  2. Anderson R. P., Menninger J. R. Tests of the ribosome editor hypothesis. III. A mutant Escherichia coli with a defective ribosome editor. Mol Gen Genet. 1987 Sep;209(2):313–318. doi: 10.1007/BF00329659. [DOI] [PubMed] [Google Scholar]
  3. Atherly A. G., Menninger J. R. Mutant E. coli strain with temperature sensitive peptidyl-transfer RNA hydrolase. Nat New Biol. 1972 Dec 20;240(103):245–246. doi: 10.1038/newbio240245a0. [DOI] [PubMed] [Google Scholar]
  4. Atherly A. G. Natural premature protein synthesis termination can be reduced in Escherichia coli by decreased translation rates. Mol Gen Genet. 1979 Oct 1;175(3):305–311. doi: 10.1007/BF00397230. [DOI] [PubMed] [Google Scholar]
  5. Blobel G., Sabatini D. D. Controlled proteolysis of nascent polypeptides in rat liver cell fractions. I. Location of the polypeptides within ribosomes. J Cell Biol. 1970 Apr;45(1):130–145. doi: 10.1083/jcb.45.1.130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bouché J. P. BEE95: a novel sequence family related to IRU/ERIC dispersed enterobacterial intergenic elements. Mol Microbiol. 1995 Dec;18(5):987–989. doi: 10.1111/j.1365-2958.1995.18050987.x. [DOI] [PubMed] [Google Scholar]
  7. Caskey C. T., Forrester W. C., Tate W., Ward C. D. Cloning of the Escherichia coli release factor 2 gene. J Bacteriol. 1984 Apr;158(1):365–368. doi: 10.1128/jb.158.1.365-368.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Caskey T., Scolnick E., Tompkins R., Goldstein J., Milman G. Peptide chain termination, codon, protein factor, and ribosomal requirements. Cold Spring Harb Symp Quant Biol. 1969;34:479–488. doi: 10.1101/sqb.1969.034.01.054. [DOI] [PubMed] [Google Scholar]
  9. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chapeville F., Yot P., Paulin D. Enzymatic hydrolysis of N-acyl-aminoacyl transfer RNAs. Cold Spring Harb Symp Quant Biol. 1969;34:493–498. doi: 10.1101/sqb.1969.034.01.055. [DOI] [PubMed] [Google Scholar]
  11. Cherepanov P. P., Wackernagel W. Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene. 1995 May 26;158(1):9–14. doi: 10.1016/0378-1119(95)00193-a. [DOI] [PubMed] [Google Scholar]
  12. Cuzin F., Kretchmer N., Greenberg R. E., Hurwitz R., Chapeville F. Enzymatic hydrolysis of N-substituted aminoacyl-tRNA. Proc Natl Acad Sci U S A. 1967 Nov;58(5):2079–2086. doi: 10.1073/pnas.58.5.2079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Frank J., Zhu J., Penczek P., Li Y., Srivastava S., Verschoor A., Radermacher M., Grassucci R., Lata R. K., Agrawal R. K. A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome. Nature. 1995 Aug 3;376(6539):441–444. doi: 10.1038/376441a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. García-Villegas M. R., De La Vega F. M., Galindo J. M., Segura M., Buckingham R. H., Guarneros G. Peptidyl-tRNA hydrolase is involved in lambda inhibition of host protein synthesis. EMBO J. 1991 Nov;10(11):3549–3555. doi: 10.1002/j.1460-2075.1991.tb04919.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grentzmann G., Brechemier-Baey D., Heurgue V., Mora L., Buckingham R. H. Localization and characterization of the gene encoding release factor RF3 in Escherichia coli. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5848–5852. doi: 10.1073/pnas.91.13.5848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gross M., Crow P., White J. The site of hydrolysis by rabbit reticulocyte peptidyl-tRNA hydrolase is the 3'-AMP terminus of susceptible tRNA substrates. J Biol Chem. 1992 Jan 25;267(3):2080–2086. [PubMed] [Google Scholar]
  18. Heurgué-Hamard V., Mora L., Guarneros G., Buckingham R. H. The growth defect in Escherichia coli deficient in peptidyl-tRNA hydrolase is due to starvation for Lys-tRNA(Lys). EMBO J. 1996 Jun 3;15(11):2826–2833. [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. 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]
  24. Jost J. P., Bock R. M. Enzymatic hydrolysis of N-substituted aminoacyl transfer ribonucleic acid in yeast. J Biol Chem. 1969 Nov 10;244(21):5866–5873. [PubMed] [Google Scholar]
  25. Jørgensen F., Kurland C. G. Processivity errors of gene expression in Escherichia coli. J Mol Biol. 1990 Oct 20;215(4):511–521. doi: 10.1016/S0022-2836(05)80164-0. [DOI] [PubMed] [Google Scholar]
  26. Kleckner N., Bender J., Gottesman S. Uses of transposons with emphasis on Tn10. Methods Enzymol. 1991;204:139–180. doi: 10.1016/0076-6879(91)04009-d. [DOI] [PubMed] [Google Scholar]
  27. Kolb V. A., Kommer A. A., Spirin A. S. Sushchestvuet li kanal dlia sinteziruemogo na ribosome peptida? Mechenie transliruiushchikh ribosom atomarnym tritiem. Dokl Akad Nauk SSSR. 1987;296(6):1497–1501. [PubMed] [Google Scholar]
  28. Komar A. A., Kommer A., Krasheninnikov I. A., Spirin A. S. Cotranslational folding of globin. J Biol Chem. 1997 Apr 18;272(16):10646–10651. doi: 10.1074/jbc.272.16.10646. [DOI] [PubMed] [Google Scholar]
  29. Kurland C. G., Ehrenberg M. Constraints on the accuracy of messenger RNA movement. Q Rev Biophys. 1985 Nov;18(4):423–450. doi: 10.1017/s0033583500005370. [DOI] [PubMed] [Google Scholar]
  30. Kössel H., RajBhandary U. L. Studies on polynucleotides. LXXXVI. Enzymic hydrolysis of N-acylaminoacyl-transfer RNA. J Mol Biol. 1968 Aug 14;35(3):539–560. doi: 10.1016/s0022-2836(68)80013-0. [DOI] [PubMed] [Google Scholar]
  31. MacDougall J., Holst-Hansen P., Mortensen K. K., Freistroffer D. V., Pavlov M. Y., Ehrenberg M., Buckingham R. H. Purification of active Escherichia coli ribosome recycling factor (RRF) from an osmo-regulated expression system. Biochimie. 1997 May;79(5):243–246. doi: 10.1016/s0300-9084(97)83511-0. [DOI] [PubMed] [Google Scholar]
  32. Malkin L. I., Rich A. Partial resistance of nascent polypeptide chains to proteolytic digestion due to ribosomal shielding. J Mol Biol. 1967 Jun 14;26(2):329–346. doi: 10.1016/0022-2836(67)90301-4. [DOI] [PubMed] [Google Scholar]
  33. Manley J. L. Synthesis and degradation of termination and premature-termination fragments of beta-galactosidase in vitro and in vivo. J Mol Biol. 1978 Nov 15;125(4):407–432. doi: 10.1016/0022-2836(78)90308-x. [DOI] [PubMed] [Google Scholar]
  34. Menninger J. R. Accumulation of peptidyl tRNA is lethal to Escherichia coli. J Bacteriol. 1979 Jan;137(1):694–696. doi: 10.1128/jb.137.1.694-696.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Menninger J. R. Peptidyl transfer RNA dissociates during protein synthesis from ribosomes of Escherichia coli. J Biol Chem. 1976 Jun 10;251(11):3392–3398. [PubMed] [Google Scholar]
  36. Menninger J. R. The accumulation as peptidyl-transfer RNA of isoaccepting transfer RNA families in Escherichia coli with temperature-sensitive peptidyl-transfer RNA hydrolase. J Biol Chem. 1978 Oct 10;253(19):6808–6813. [PubMed] [Google Scholar]
  37. Mikuni O., Ito K., Moffat J., Matsumura K., McCaughan K., Nobukuni T., Tate W., Nakamura Y. Identification of the prfC gene, which encodes peptide-chain-release factor 3 of Escherichia coli. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5798–5802. doi: 10.1073/pnas.91.13.5798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Milligan R. A., Unwin P. N. Location of exit channel for nascent protein in 80S ribosome. Nature. 1986 Feb 20;319(6055):693–695. doi: 10.1038/319693a0. [DOI] [PubMed] [Google Scholar]
  40. Milman G., Goldstein J., Scolnick E., Caskey T. Peptide chain termination. 3. Stimulation of in vitro termination. Proc Natl Acad Sci U S A. 1969 May;63(1):183–190. doi: 10.1073/pnas.63.1.183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pavlov M. Y., Freistroffer D. V., Heurgué-Hamard V., Buckingham R. H., Ehrenberg M. Release factor RF3 abolishes competition between release factor RF1 and ribosome recycling factor (RRF) for a ribosome binding site. J Mol Biol. 1997 Oct 24;273(2):389–401. doi: 10.1006/jmbi.1997.1324. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Prokaryotic and eukaryotic translation factors. Ad Hoc Nomenclature Subcommittee Report. Biochimie. 1996;78(11-12):1119–1122. [PubMed] [Google Scholar]
  44. Ryabova L. A., Selivanova O. M., Baranov V. I., Vasiliev V. D., Spirin A. S. Does the channel for nascent peptide exist inside the ribosome? Immune electron microscopy study. FEBS Lett. 1988 Jan 4;226(2):255–260. doi: 10.1016/0014-5793(88)81434-0. [DOI] [PubMed] [Google Scholar]
  45. Régnier P., Grunberg-Manago M. Cleavage by RNase III in the transcripts of the met Y-nus-A-infB operon of Escherichia coli releases the tRNA and initiates the decay of the downstream mRNA. J Mol Biol. 1989 Nov 20;210(2):293–302. doi: 10.1016/0022-2836(89)90331-8. [DOI] [PubMed] [Google Scholar]
  46. Régnier P., Portier C. Initiation, attenuation and RNase III processing of transcripts from the Escherichia coli operon encoding ribosomal protein S15 and polynucleotide phosphorylase. J Mol Biol. 1986 Jan 5;187(1):23–32. doi: 10.1016/0022-2836(86)90403-1. [DOI] [PubMed] [Google Scholar]
  47. Scolnick E., Tompkins R., Caskey T., Nirenberg M. Release factors differing in specificity for terminator codons. Proc Natl Acad Sci U S A. 1968 Oct;61(2):768–774. doi: 10.1073/pnas.61.2.768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Shevell D. E., Abou-Zamzam A. M., Demple B., Walker G. C. Construction of an Escherichia coli K-12 ada deletion by gene replacement in a recD strain reveals a second methyltransferase that repairs alkylated DNA. J Bacteriol. 1988 Jul;170(7):3294–3296. doi: 10.1128/jb.170.7.3294-3296.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  51. Wang R. F., Kushner S. R. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene. 1991 Apr;100:195–199. [PubMed] [Google Scholar]
  52. Wang S., Sakai H., Wiedmann M. NAC covers ribosome-associated nascent chains thereby forming a protective environment for regions of nascent chains just emerging from the peptidyl transferase center. J Cell Biol. 1995 Aug;130(3):519–528. doi: 10.1083/jcb.130.3.519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Weiss R. B., Murphy J. P., Gallant J. A. Genetic screen for cloned release factor genes. J Bacteriol. 1984 Apr;158(1):362–364. doi: 10.1128/jb.158.1.362-364.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yanofsky C. Mutations affecting tRNATrp and its charging and their effect on regulation of transcription termination at the attenuator of the tryptophan operon. J Mol Biol. 1977 Jul 15;113(4):663–677. doi: 10.1016/0022-2836(77)90229-7. [DOI] [PubMed] [Google Scholar]

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