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. 1994 Jun 21;91(13):5798–5802. doi: 10.1073/pnas.91.13.5798

Identification of the prfC gene, which encodes peptide-chain-release factor 3 of Escherichia coli.

O Mikuni 1, K Ito 1, J Moffat 1, K Matsumura 1, K McCaughan 1, T Nobukuni 1, W Tate 1, Y Nakamura 1
PMCID: PMC44084  PMID: 8016068

Abstract

The termination of protein synthesis in bacteria requires two codon-specific polypeptide release factors, RF-1 and RF-2. A third factor, RF-3, which stimulates the RF-1 and RF-2 activities, was originally identified in Escherichia coli, but it has received little attention since the 1970s. To search for the gene encoding RF-3, we selected nonsense-suppressor mutations by random insertion mutagenesis on the assumption that a loss of function of RF-3 would lead to misreading of stop signals. One of these mutations, named tos-1 (for transposon-induced opal suppressor), mapped to the 99.2 min region on the E. coli chromosome and suppressed all three stop codons. Complementation studies and analyses of the DNA and protein sequences revealed that the tos gene encodes a 59,442-Da protein, with sequence homology to elongation factor EF-G, including G-domain motifs, and that the tos-1 insertion eliminated the C-terminal one-fifth of the protein. Extracts containing the overproduced Tos protein markedly increased the formation of ribosomal termination complexes and stimulated the RF-1 or RF-2 activity in the codon-dependent in vitro termination assay. The stimulation was significantly reduced by GTP, GDP, and the beta,gamma-methylene analog of GTP, but not by GMP. These results fit perfectly with those described in the original publications on RF-3, and the tos gene has therefore been designated prfC. A completely null prfC mutation made by reverse genetics affected the cell growth under the limited set of physiological and strain conditions.

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Selected References

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  1. Capecchi M. R., Klein H. A. Characterization of three proteins involved in polypeptide chain termination. Cold Spring Harb Symp Quant Biol. 1969;34:469–477. doi: 10.1101/sqb.1969.034.01.053. [DOI] [PubMed] [Google Scholar]
  2. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Connolly D. M., Winkler M. E. Structure of Escherichia coli K-12 miaA and characterization of the mutator phenotype caused by miaA insertion mutations. J Bacteriol. 1991 Mar;173(5):1711–1721. doi: 10.1128/jb.173.5.1711-1721.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Craigen W. J., Lee C. C., Caskey C. T. Recent advances in peptide chain termination. Mol Microbiol. 1990 Jun;4(6):861–865. doi: 10.1111/j.1365-2958.1990.tb00658.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Elliott T., Wang X. Salmonella typhimurium prfA mutants defective in release factor 1. J Bacteriol. 1991 Jul;173(13):4144–4154. doi: 10.1128/jb.173.13.4144-4154.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Groisman E. A., Casadaban M. J. Mini-mu bacteriophage with plasmid replicons for in vivo cloning and lac gene fusing. J Bacteriol. 1986 Oct;168(1):357–364. doi: 10.1128/jb.168.1.357-364.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Göringer H. U., Hijazi K. A., Murgola E. J., Dahlberg A. E. Mutations in 16S rRNA that affect UGA (stop codon)-directed translation termination. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6603–6607. doi: 10.1073/pnas.88.15.6603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ito K., Kawakami K., Nakamura Y. Multiple control of Escherichia coli lysyl-tRNA synthetase expression involves a transcriptional repressor and a translational enhancer element. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):302–306. doi: 10.1073/pnas.90.1.302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Kawakami K., Jönsson Y. H., Björk G. R., Ikeda H., Nakamura Y. Chromosomal location and structure of the operon encoding peptide-chain-release factor 2 of Escherichia coli. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5620–5624. doi: 10.1073/pnas.85.15.5620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kawakami K., Nakamura Y. Autogenous suppression of an opal mutation in the gene encoding peptide chain release factor 2. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8432–8436. doi: 10.1073/pnas.87.21.8432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  16. Martinez E., Bartolomé B., de la Cruz F. pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids. Gene. 1988 Aug 15;68(1):159–162. doi: 10.1016/0378-1119(88)90608-7. [DOI] [PubMed] [Google Scholar]
  17. McCaughan K. K., Ward C. D., Trotman C. N., Tate W. P. The ribosomal binding domain for the bacterial release factors RF-1, RF-2 and RF-3. FEBS Lett. 1984 Sep 17;175(1):90–94. doi: 10.1016/0014-5793(84)80576-1. [DOI] [PubMed] [Google Scholar]
  18. Murgola E. J., Hijazi K. A. Selection for new codons corresponding to position 234 of the tryptophan synthetase alpha chain of Escherichia coli. Mol Gen Genet. 1983;191(1):132–137. doi: 10.1007/BF00330900. [DOI] [PubMed] [Google Scholar]
  19. Roesser J. R., Nakamura Y., Yanofsky C. Regulation of basal level expression of the tryptophan operon of Escherichia coli. J Biol Chem. 1989 Jul 25;264(21):12284–12288. [PubMed] [Google Scholar]
  20. 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]
  21. Rydén S. M., Isaksson L. A. A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for some tRNA nonsense suppressors. Mol Gen Genet. 1984;193(1):38–45. doi: 10.1007/BF00327411. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Singer M., Baker T. A., Schnitzler G., Deischel S. M., Goel M., Dove W., Jaacks K. J., Grossman A. D., Erickson J. W., Gross C. A. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol Rev. 1989 Mar;53(1):1–24. doi: 10.1128/mr.53.1.1-24.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Soll L. Mutational alterations of tryptophan-specific transfer RNA that generate translation suppressors of the UAA, UAG and UGA nonsense codons. J Mol Biol. 1974 Jun 25;86(2):233–243. doi: 10.1016/0022-2836(74)90015-1. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  27. Way J. C., Davis M. A., Morisato D., Roberts D. E., Kleckner N. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene. 1984 Dec;32(3):369–379. doi: 10.1016/0378-1119(84)90012-x. [DOI] [PubMed] [Google Scholar]
  28. 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]

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