1887

Abstract

Peptidyl-tRNA hydrolase (Pth) in is required to recycle tRNA molecules that dissociate from the ribosome as peptidyl-tRNA during protein synthesis. At non-permissive temperatures, strains with a thermosensitive mutation affecting the enzyme accumulate peptidyl-tRNA, cease protein synthesis and die. The rate of reversion of this mutation to thermoresistance varies widely according to the genetic background of the cell and the temperature of selection; under certain conditions, reversion can occur at rates approaching 10 per cell per generation. In such revertants, a chromosomal gene can be replaced by an inactivated gene, restoring thermosensitive growth in most cases. PCR amplification experiments and Southern blots show the presence of both normal and inactivated copies of the gene, demonstrating that gene duplication has occurred in the revertants. Estimation of intracellular peptidyl-tRNA hydrolase by Western blotting confirms this explanation of the mechanism of high-frequency reversion to thermoresistance.

Keyword(s): ribosome , termination and translation
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2001-06-01
2019-12-06
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References

  1. Anderson, P. & Roth, J. ( 1981; ). Spontaneous tandem genetic duplications in Salmonella typhimurium arise by unequal recombination between rRNA (rrn) cistrons. Proc Natl Acad Sci USA 78, 3113-3117.[CrossRef]
    [Google Scholar]
  2. Atherly, A. G. & Menninger, J. R. ( 1972; ). Mutant Escherichia coli strain with temperature sensitive peptidyl-transfer RNA hydrolase. Nature 240, 245-246.[CrossRef]
    [Google Scholar]
  3. Bachellier, S., Gilson, E., Hofnung, M. & Hill, C. W. (1966). Repeated sequences. In Escherichia coli and Salmonella: Cellular and Molecular Biology. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
  4. Blomfield, I. C., Vaughn, V., Rest, R. F. & Eisenstein, B. I. ( 1991; ). Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol Microbiol 5, 1447-1457.[CrossRef]
    [Google Scholar]
  5. Coulondre, C. & Miller, J. H. ( 1977; ). Genetic studies of the lac repressor III: additional correlation of mutational sites with specific aminoacid residues. J Mol Biol 117, 525-575.[CrossRef]
    [Google Scholar]
  6. Cruz-Vera, L. R., Toledo, I., Hernandez-Sanchez, J. & Guarneros, G. ( 2000; ). Molecular basis for the temperature sensitivity of Escherichia coli pth(Ts). J Bacteriol 182, 1523-1528.[CrossRef]
    [Google Scholar]
  7. De La Vega, F. M., Galindo, J. M., Old, I. G. & Guarneros, G. ( 1996; ). Microbial genes homologous to the peptidyl-tRNA hydrolase-encoding gene of Escherichia coli. Gene 169, 97-100.[CrossRef]
    [Google Scholar]
  8. Dinçbas, V., Heurgué-Hamard, V., Buckingham, R. H., Karimi, R. & Ehrenberg, M. ( 1999; ). Shutdown in protein synthesis due to the expression of mini-genes in bacteria. J Mol Biol 291, 745-759.[CrossRef]
    [Google Scholar]
  9. Dinçbas-Renqvist, V., Engström, Å., Mora, L., Heurgué-Hamard, V., Buckingham, R. H. & Ehrenberg, M. ( 2000; ). A post-translational modification in the GGQ motif of RF2 from E. coli stimulates termination of translation. EMBO J 19, 6900-6907.[CrossRef]
    [Google Scholar]
  10. Drake, J. W. ( 1969; ). Comparative rates of spontaneous mutation. Nature 221, 1132.[CrossRef]
    [Google Scholar]
  11. Dutka, S., Meinnel, T., Lazennec, C., Mechulam, Y. & Blanquet, S. ( 1993; ). Role of the 1–72 base pair in tRNAs for the activity of Escherichia coli peptidyl-tRNA hydrolase. Nucleic Acids Res 21, 4025-4030.[CrossRef]
    [Google Scholar]
  12. Folk, W. R. & Berg, P. ( 1971; ). Duplication of the structural gene for glycyl-transfer RNA synthetase in Escherichia coli. J Mol Biol 58, 595-610.[CrossRef]
    [Google Scholar]
  13. Gross, M., Crow, P. & White, J. ( 1992a; ). The site of hydrolysis by rabbit reticulocyte peptidyl-tRNA hydrolase is the 3′-AMP terminus of susceptible tRNA substrates. J Biol Chem 267, 2080-2086.
    [Google Scholar]
  14. Gross, M., Starn, T. K., Rundquist, C., Crow, P., White, J., Olin, A. & Wagner, T. ( 1992b; ). Purification and initial characterization of peptidyl-tRNA hydrolase from rabbit reticulocytes. J Biol Chem 267, 2073-2079.
    [Google Scholar]
  15. Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. ( 1995; ). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177, 4121-4130.
    [Google Scholar]
  16. Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, P. & Kushner, S. R. ( 1989; ). New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol 171, 4617-4622.
    [Google Scholar]
  17. Heurgué-Hamard, V., Mora, L., Guarneros, G. & Buckingham, R. H. ( 1996; ). The growth defect in E. coli deficient in peptidyl-tRNA hydrolase is due to starvation for Lys-tRNALys. EMBO J 15, 2826-2833.
    [Google Scholar]
  18. Heurgué-Hamard, V., Karimi, R., Mora, L., MacDougall, J., Leboeuf, C., Grentzmann, G., Ehrenberg, M. & Buckingham, R. H. ( 1998; ). Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome. EMBO J 17, 808-816.[CrossRef]
    [Google Scholar]
  19. Hughes, D. ( 1999; ). The impact of homologous recombination on genome organisation and stability. In Organisation of the Prokaryotic Genome , pp. 109-128. Edited by R. L. Charlebois. Washington, DC:American Society for Microbiology.
  20. Jessop, A. P. & Clugston, C. ( 1985; ). Amplification of the argF region in strain HfrP4X of E. coli K-12. Mol Gen Genet 201, 347-350.[CrossRef]
    [Google Scholar]
  21. Jørgensen, F. & Kurland, C. G. ( 1990; ). Processivity errors of gene expression in Escherichia coli. J Mol Biol 215, 511-521.[CrossRef]
    [Google Scholar]
  22. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[CrossRef]
    [Google Scholar]
  23. Lin, R. J., Capage, M. & Hill, C. W. ( 1984; ). A repetitive DNA sequence, rhs, responsible for duplications within the Escherichia coli K-12 chromosome. J Mol Biol 177, 1-18.[CrossRef]
    [Google Scholar]
  24. Luria, S. E. & Delbrück, M. ( 1943; ). Mutations of bacteria from virus sensitivity to virus resistance. Genetics 28, 491-511.
    [Google Scholar]
  25. Manley, J. L. ( 1978; ). Synthesis and degradation of termination and premature termination fragments of beta-galactosidase in vitro. J Mol Biol 125, 407-432.[CrossRef]
    [Google Scholar]
  26. Menez, J., Heurgue-Hamard, V. & Buckingham, R. H. ( 2000; ). Sequestration of specific tRNA species cognate to the last sense codon of an overproduced gratuitous protein. Nucleic Acids Res 28, 4733-4741.[CrossRef]
    [Google Scholar]
  27. Menninger, J. R. ( 1978; ). The accumulation as peptidyl-transfer RNA of isoaccepting transfer RNA families in E. coli with temperature-sensitive peptidyl-transfer RNA hydrolase. J Biol Chem 253, 6808-6813.
    [Google Scholar]
  28. Miller, J. H. (1992). A Short Course in Bacterial Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  29. Ouzounis, C., Bork, P., Casari, G. & Sander, C. ( 1995; ). New protein functions in yeast chromosome VIII. Protein Sci 4, 2424-2428.[CrossRef]
    [Google Scholar]
  30. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  31. Schmitt, E., Mechulam, Y., Fromant, M., Plateau, P. & Blanquet, S. ( 1997; ). Crystal structure at 1·2Å resolution and active site mapping of Escherichia coli peptidyl-tRNA hydrolase. EMBO J 16, 4760-4769.[CrossRef]
    [Google Scholar]
  32. Shyamala, V., Schneider, E. & Ames, G. F. ( 1990; ). Tandem chromosomal duplications: role of REP sequences in the recombination event at the join-point. EMBO J 9, 939-946.
    [Google Scholar]
  33. Tlsty, T. D., Albertini, A. M. & Miller, J. H. ( 1984; ). Gene amplification in the lac region of E. coli. Cell 37, 217-224.[CrossRef]
    [Google Scholar]
  34. Vogel, Z., Zamir, A. & Elson, D. ( 1968; ). On the specificity and stability of an enzyme that hydrolyses N-substituted aminoacyl-transfer RNAs. Proc Natl Acad Sci USA 61, 701-707.[CrossRef]
    [Google Scholar]
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