Abstract
The existence of a conditional lethal temperature-sensitive mutant affecting peptidyl-tRNA hydrolase in Escherichia coli suggests that this enzyme is essential to cell survival. We report here the isolation of both chromosomal and multicopy suppressors of this mutant in pth, the gene encoding the hydrolase. In one case, the cloned gene responsible for suppression is shown to be lysV, one of three genes encoding the unique lysine acceptor tRNA; 10 other cloned tRNA genes are without effect. Overexpression of lysV leading to a 2- to 3-fold increase in tRNA(Lys) concentration overcomes the shortage of peptidyl-tRNA hydrolase activity in the cell at non-permissive temperature. Conversely, in pth, supN double mutants, where the tRNA(Lys) concentration is reduced due to the conversion of lysV to an ochre suppressor (supN), the thermosensitivity of the initial pth mutant becomes accentuated. Thus, cells carrying both mutations show practically no growth at 39 degrees C, a temperature at which the pth mutant grows almost normally. Growth of the double mutant is restored by the expression of lysV from a plasmid. These results indicate that the limitation of growth in mutants of E.coli deficient in Pth is due to the sequestration of tRNA(Lys) as peptidyl-tRNA. This is consistent with previous observations that this tRNA is particularly prone to premature dissociation from the ribosome.
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- 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]
- 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]
- 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]
- Atherly A. G. Peptidyl-transfer RNA hydrolase prevents inhibition of protein synthesis initiation. Nature. 1978 Oct 26;275(5682):769–769. doi: 10.1038/275769a0. [DOI] [PubMed] [Google Scholar]
- Atkins J. F., Elseviers D., Gorini L. Low activity of -galactosidase in frameshift mutants of Escherichia coli. Proc Natl Acad Sci U S A. 1972 May;69(5):1192–1195. doi: 10.1073/pnas.69.5.1192. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Aulin M. R., Hughes D. Overproduction of release factor reduces spontaneous frameshifting and frameshift suppression by mutant elongation factor Tu. J Bacteriol. 1990 Dec;172(12):6721–6726. doi: 10.1128/jb.172.12.6721-6726.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brun Y. V., Breton R., Lanouette P., Lapointe J. Precise mapping and comparison of two evolutionarily related regions of the Escherichia coli K-12 chromosome. Evolution of valU and lysT from an ancestral tRNA operon. J Mol Biol. 1990 Aug 20;214(4):825–843. doi: 10.1016/0022-2836(90)90339-N. [DOI] [PubMed] [Google Scholar]
- Brun Y. V., Sanfaçon H., Breton R., Lapointe J. Closely spaced and divergent promoters for an aminoacyl-tRNA synthetase gene and a tRNA operon in Escherichia coli. Transcriptional and post-transcriptional regulation of gltX, valU and alaW. J Mol Biol. 1990 Aug 20;214(4):845–864. doi: 10.1016/0022-2836(90)90340-R. [DOI] [PubMed] [Google Scholar]
- COX R. A., LITTAUER U. Z. Ribonucleic acid from Escherichia coli. III. The influence of ionic strength and temperature on hydrodynamic and optical properties. Biochim Biophys Acta. 1962 Aug 20;61:197–208. [PubMed] [Google Scholar]
- Caillet J., Plumbridge J. A., Springer M. Evidence that pheV, a gene for tRNAPhe of E. coli is transcribed from tandem promoters. Nucleic Acids Res. 1985 May 24;13(10):3699–3710. doi: 10.1093/nar/13.10.3699. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Cummings H. S., Sands J. F., Fraser J., Hershey J. W. Characterization and expression of a gene encoding serine tRNA5 from Escherichia coli. Biochimie. 1994;76(1):83–87. doi: 10.1016/0300-9084(94)90067-1. [DOI] [PubMed] [Google Scholar]
- Delamarche C., Vacher J., Buckingham R. H. Mutants affecting tRNA(Phe) from Escherichia coli. Studies of the suppression of thermosensitive phenylalanyl-tRNA synthetase. Eur J Biochem. 1987 Oct 15;168(2):365–369. doi: 10.1111/j.1432-1033.1987.tb13428.x. [DOI] [PubMed] [Google Scholar]
- Dong H., Kurland C. G. Ribosome mutants with altered accuracy translate with reduced processivity. J Mol Biol. 1995 May 5;248(3):551–561. doi: 10.1006/jmbi.1995.0242. [DOI] [PubMed] [Google Scholar]
- Emilsson V., Kurland C. G. Growth rate dependence of transfer RNA abundance in Escherichia coli. EMBO J. 1990 Dec;9(13):4359–4366. doi: 10.1002/j.1460-2075.1990.tb07885.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Emilsson V., Näslund A. K., Kurland C. G. Growth-rate-dependent accumulation of twelve tRNA species in Escherichia coli. J Mol Biol. 1993 Mar 20;230(2):483–491. doi: 10.1006/jmbi.1993.1165. [DOI] [PubMed] [Google Scholar]
- Fayet O., Louarn J. M., Georgopoulos C. Suppression of the Escherichia coli dnaA46 mutation by amplification of the groES and groEL genes. Mol Gen Genet. 1986 Mar;202(3):435–445. doi: 10.1007/BF00333274. [DOI] [PubMed] [Google Scholar]
- 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]
- Gordon C. L., Sather S. K., Casjens S., King J. Selective in vivo rescue by GroEL/ES of thermolabile folding intermediates to phage P22 structural proteins. J Biol Chem. 1994 Nov 11;269(45):27941–27951. [PubMed] [Google Scholar]
- Haenni A. L., Chapeville F. The behaviour of acetylphenylalanyl soluble ribonucleic acid in polyphenylalanine synthesis. Biochim Biophys Acta. 1966 Jan 18;114(1):135–148. doi: 10.1016/0005-2787(66)90261-9. [DOI] [PubMed] [Google Scholar]
- Henderson D., Weil J. A mutant of Escherichia coli that prevents growth of phage lambda and is bypassed by lambda mutants in a nonessential region of the genome. Virology. 1976 Jun;71(2):546–559. doi: 10.1016/0042-6822(76)90380-9. [DOI] [PubMed] [Google Scholar]
- Heurgué-Hamard V., Mora L., Buckingham R. H. Rapid and precise chromosomal mapping of genomic probes in Escherichia coli using the digital physical map. Nucleic Acids Res. 1995 Jul 25;23(14):2801–2802. doi: 10.1093/nar/23.14.2801-a. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jørgensen F., Adamski F. M., Tate W. P., Kurland C. G. Release factor-dependent false stops are infrequent in Escherichia coli. J Mol Biol. 1993 Mar 5;230(1):41–50. doi: 10.1006/jmbi.1993.1124. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Komine Y., Inokuchi H. Importance of the G27-A43 mismatch at the anticodon stem of Escherichia coli tRNA(Thr2). FEBS Lett. 1990 Oct 15;272(1-2):55–57. doi: 10.1016/0014-5793(90)80447-q. [DOI] [PubMed] [Google Scholar]
- 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]
- Maloy S. R., Nunn W. D. Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol. 1981 Feb;145(2):1110–1111. doi: 10.1128/jb.145.2.1110-1111.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Marchuk D., Drumm M., Saulino A., Collins F. S. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 1991 Mar 11;19(5):1154–1154. doi: 10.1093/nar/19.5.1154. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Menninger J. R. Ribosome editing and the error catastrophe hypothesis of cellular aging. Mech Ageing Dev. 1977 Mar-Apr;6(2):131–142. doi: 10.1016/0047-6374(77)90014-8. [DOI] [PubMed] [Google Scholar]
- 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]
- Menninger J. R., Walker C., Tan P. F. Studies on the metabolic role of peptidyl-tRNA hydrolase. I. Properties of a mutant E. coli with temperature-sensitive peptidyl-tRNA hydrolase. Mol Gen Genet. 1973 Mar 19;121(4):307–324. doi: 10.1007/BF00433230. [DOI] [PubMed] [Google Scholar]
- Mierendorf R. C., Pfeffer D. Direct sequencing of denatured plasmid DNA. Methods Enzymol. 1987;152:556–562. doi: 10.1016/0076-6879(87)52061-4. [DOI] [PubMed] [Google Scholar]
- O'Connor M., Willis N. M., Bossi L., Gesteland R. F., Atkins J. F. Functional tRNAs with altered 3' ends. EMBO J. 1993 Jun;12(6):2559–2566. doi: 10.1002/j.1460-2075.1993.tb05911.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Smith D., Yarus M. Transfer RNA structure and coding specificity. I. Evidence that a D-arm mutation reduces tRNA dissociation from the ribosome. J Mol Biol. 1989 Apr 5;206(3):489–501. doi: 10.1016/0022-2836(89)90496-8. [DOI] [PubMed] [Google Scholar]
- Springer M., Graffe M., Dondon J., Grunberg-Manago M. tRNA-like structures and gene regulation at the translational level: a case of molecular mimicry in Escherichia coli. EMBO J. 1989 Aug;8(8):2417–2424. doi: 10.1002/j.1460-2075.1989.tb08372.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tsuchihashi Z., Brown P. O. Sequence requirements for efficient translational frameshifting in the Escherichia coli dnaX gene and the role of an unstable interaction between tRNA(Lys) and an AAG lysine codon. Genes Dev. 1992 Mar;6(3):511–519. doi: 10.1101/gad.6.3.511. [DOI] [PubMed] [Google Scholar]
- Uemura H., Thorbjarnardóttir S., Gamulin V., Yano J., Andrésson O. S., Söll D., Eggertsson G. supN ochre suppressor gene in Escherichia coli codes for tRNALys. J Bacteriol. 1985 Sep;163(3):1288–1289. doi: 10.1128/jb.163.3.1288-1289.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Van Dyk T. K., Gatenby A. A., LaRossa R. A. Demonstration by genetic suppression of interaction of GroE products with many proteins. Nature. 1989 Nov 23;342(6248):451–453. doi: 10.1038/342451a0. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Zeilstra-Ryalls J., Fayet O., Baird L., Georgopoulos C. Sequence analysis and phenotypic characterization of groEL mutations that block lambda and T4 bacteriophage growth. J Bacteriol. 1993 Feb;175(4):1134–1143. doi: 10.1128/jb.175.4.1134-1143.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]