Molecular basis for the temperature sensitivity of Escherichia coli pth(Ts)

The gene pth, encoding peptidyl-tRNA hydrolase (Pth), is essential for prot ein synthesis and viability of Escherichia coli. Two pth mutants have been studied in depth: a pth(Ts) mutant isolated as temperature sensitive and a pth(rap) mutant selected as nonpermissive for bacteriophage lambda vegetati ve growth. Here we show that each mutant protein is defective in a differen t way. The Pth(Ts) protein was very unstable in vivo, both at 43 degrees C and at permissive temperatures, but its specific activity was comparable to that of the wild-type enzyme, Pth(wt). Conversely, the mutant Pth(rap) pro tein had the same stability as Pth(wt), but its specific activity was low. The thermosensitivity of the pth(Ts) mutant, presumably, ensues after Pth(T s) protein levels are reduced at 43 degrees C. Conditions that increased th e cellular Pth(Ts) concentration, a rise in gene copy number or diminished protein degradation, allowed cell growth at a nonpermissive temperature. An tibiotic-mediated inhibition of mRNA and protein synthesis, but not of pept idyl-tRNA drop-off, reduced pth(Ts) cell viability even at a permissive tem perature. Based on these results, we suggest that Pth(Ts) protein, being un stable in vivo, supports cell viability only if its concentration is mainta ined above a threshold that allows general protein synthesis.