Peptidyl-tRNA hydrolase (Pth) in Escherichia coli 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 10−3 per cell per generation. In such revertants, a chromosomal pth 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.
AndersonP.,
RothJ.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]
BachellierS., GilsonE., HofnungM.,
HillC. W.others1966; Repeated sequences. In Escherichia coli and Salmonella: Cellular and Molecular Biology Edited by
NeidhardtF. C.
Washington, DC: American Society for Microbiology;
CoulondreC.,
MillerJ. 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]
Cruz-VeraL. R., ToledoI., Hernandez-SanchezJ.,
GuarnerosG.2000; Molecular basis for the temperature sensitivity of Escherichia coli pth (Ts). J Bacteriol 182:1523–1528[CrossRef]
De La VegaF. M., GalindoJ. M., OldI. G.,
GuarnerosG.1996; Microbial genes homologous to the peptidyl-tRNA hydrolase-encoding gene of Escherichia coli.. Gene 169:97–100[CrossRef]
DinçbasV.,
Heurgué-HamardV., BuckinghamR. H., KarimiR.,
EhrenbergM.1999; Shutdown in protein synthesis due to the expression of mini-genes in bacteria. J Mol Biol 291:745–759[CrossRef]
Dinçbas-RenqvistV.,
EngströmÅ., MoraL., BuckinghamR. H.,
EhrenbergM., Heurgué-HamardV.2000; A post-translational modification in the GGQ motif of RF2 from E. coli stimulates termination of translation. EMBO J 19:6900–6907[CrossRef]
DutkaS., MeinnelT., LazennecC., MechulamY.,
BlanquetS.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]
GrossM., CrowP.,
WhiteJ.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
HamiltonC. M., AldeaM., WashburnB. K., BabitzkeP.,
KushnerS. R.1989; New method for generating deletions and gene replacements in Escherichia coli. . J Bacteriol 171:4617–4622
Heurgué-HamardV., MoraL., GuarnerosG.,
BuckinghamR. 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
Heurgué-HamardV., KarimiR., MoraL., MacDougallJ., LeboeufC., GrentzmannG., EhrenbergM.,
BuckinghamR. 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]
HughesD.1999; The impact of homologous recombination on genome organisation and stability. In Organisation of the Prokaryotic Genome pp 109–128 Edited by
CharleboisR. L.
Washington, DC: American Society for Microbiology;
LinR. J., CapageM.,
HillC. W.1984; A repetitive DNA sequence, rhs, responsible for duplications within the Escherichia coli K-12 chromosome. J Mol Biol 177:1–18[CrossRef]
ManleyJ. L.1978; Synthesis and degradation of termination and premature termination fragments of beta-galactosidase in vitro. . J Mol Biol 125:407–432[CrossRef]
MenezJ., Heurgue-HamardV.,
BuckinghamR. 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]
MenningerJ. 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
SchmittE., MechulamY., FromantM., PlateauP.,
BlanquetS.1997; Crystal structure at 1·2Å resolution and active site mapping of Escherichia coli peptidyl-tRNA hydrolase. EMBO J 16:4760–4769[CrossRef]
ShyamalaV., SchneiderE.,
AmesG. F.1990; Tandem chromosomal duplications: role of REP sequences in the recombination event at the join-point. EMBO J 9:939–946
VogelZ., ZamirA.,
ElsonD.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]