1564–1570 Nucleic Acids Research, 2006, Vol. 34, No. 5 doi:10.1093/nar/gkl046 Lys Excess of charged tRNA maintains low levels of peptidyl-tRNA hydrolase in pth(Ts) mutants at a non-permissive temperature Serafin Vivanco-Dom´ınguez1, Luis Rogelio Cruz-Vera2 and Gabriel Guarneros1,* 1Departamento de Gene´ tica y Biolog´ıa Molecular, Centro de Investigacio´ n y de Estudios Avanzados del IPN, Apartado Postal 14-740, 07000 Me´ xico D.F., Me´ xico and 2Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA Received January 9, 2006; Revised February 11, 2006; Accepted February 21, 2006 ABSTRACT Cellular changes have been monitored during the suppression, mediated by the overproduction of tRNALys, of thermosensitivity in Escherichia coli strain AA7852 carrying a mutation in peptidyl-tRNA hydrolase (Pth) encoded by the pth(Ts) gene. The presence in AA7852 cells of a plasmid bearing lysV gene helped to maintain low levels of the unstable Pth(Ts) protein and to preserve the viability of the mutant line at 41 C whereas plasmids bearing other tRNA genes were ineffective. At 32 C the excess of tRNALys did not alter the percentages of the free-, charged- or peptidyl-tRNALys species compared with those found in strains that did not overproduce tRNALys. At 41 C, however, despite increases in the level of peptidyl-tRNALys, the excess tRNALys helped to maintain the concentration of charged-tRNALys at a level comparable with that found in non-overproducer cells grown at a permissive temperature. In addition, the excess tRNALys at 41 C provoked a reduction in the concentrations of various peptidyl-tRNAs, which normally accumulate in pth(Ts) cells, and a proportional increase in the concentrations of the corresponding aminoacyl-tRNAs. The possible mechanism of rescue due to the overexpression of tRNALys and the causes of tRNALys starvation in pth(Ts) strains grown at non-permissive tempera- tures are considered. INTRODUCTION Peptidyl-tRNA hydrolase (Pth) hydrolyses the peptidyl- tRNAs (p-tRNAs) that are prematurely dissociated from ribosomes, thus regenerating free aminoacylable tRNAs nec- essary for new rounds of protein synthesis. In Escherichia coli this enzyme, which is essential for the synthesis of proteins and for the viability of the bacterium, is encoded by the gene pth. The mutant gene pth(Ts) encodes a mature protein with a single amino acid substitution at residue 100 (Gly to Asp) giving rise to a temperature sensitive phenotype (1,2). Whilst Pth(Ts) protein is quite unstable in vivo even at permissive temperatures, an increase in temperature to 43 C causes the protein concentration to fall rapidly to undetectable levels within 10 min (3). Indeed, soon after a shift to temperatures higher than 39 C, p-tRNAs accumulate, protein synthesis is arrested, and the mutant pth(Ts) cells die (4,5). A number of different mechanisms are known by which the thermosensitivity of a pth(Ts) strain may be suppressed, mediated either through a variation in the level of Pth(Ts) protein or an alteration in the metabolism of p-tRNA in the cell. The ﬁrst group includes the over expression of pth(Ts) per se (3). Thus, chromosomal duplication of pth(Ts) has been shown to increase the level of Pth(Ts) protein and to maintain the viability of the mutant (6). Moreover, the over expression of the chaperonins GroEL and GroES, and a defect in the dnaJ gene, can stabilize the Pth(Ts) protein and therefore enhance its level (7,3). The second group of suppressors comprises factors involved in the dissociation of p-tRNA from ribosomes. Thus, mutations in the factors RRF and RF3 restore the growth of pth(Ts) cells through a reduction in drop-off (8), whilst the overproduction of tmRNA (10Sa RNA) suppresses thermosensitivity by reducing stalled p-tRNA–ribosome complexes (9). Of particular note is the ﬁnding of Heurgue´-Hamard et al. (7) that the overproduction of tRNALys, but not of certain other tRNAs, is also able to suppress the Pth(Ts) phenotype. It has been shown that pth(Ts) mutant cells, when incubated at 40 C, accumulate different families of isoacceptors as p-tRNAs at various rates, but that p-tRNALys accumulates most rapidly (4). Overproduction of tRNALys seems to *To whom correspondence should be addressed. Tel: +52 55 5061 3338; Fax: +52 55 5061 3392; Email: gguarner@cinvestav.mx The Author 2006. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. 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