The relationship between translational control and mRNA degradation for the Escherichia coli threonyl-tRNA synthetase gene

Expression of thrS, the gene encoding Escherichia coli threonyl-tRNA synthe tase, is negatively autoregulated at the translational level. Regulation is due to the binding of threonyl-tRNA synthetase to its own mRNA at a site c alled the operator, located immediately upstream of the initiation codon. T he present work investigates the relationship between regulation and mRNA d egradation. We show that two regulatory mutations, which increase thrS expr ession, cause an increase in the steady-state mRNA concentration. Unexpecte dly, however, the half-life of thrS mRNA in the derepressed mutants is equa l to that of the wild-type, indicating that mRNA stability is independent o f the repression level. All our results can be explained if one assumes tha t thrS mRNA is either fully translated or immediately degraded. The immedia tely degraded RNAs are never detected due to their extremely short half-liv es, while the fully translated messengers share the same half-lives, irresp ective of the mutations. The increase in the steady-state level of thrS mRN A in the derepressed mutants is simply explained by an increase in the popu lation of translated molecules, i.e. those never bound by the repressor, Th rRS. Despite this peculiarity, thrS mRNA degradation seems to follow the cl assical degradation pathway. Its stability is increased in a strain defecti ve for RNase E, indicating that an endonucleolytic cleavage by this enzyme is the rate-limiting process in degradation. We also observe an accumulatio n of small fragments corresponding to the 5 ' end of the message in a strai n defective for polynucleotide phosphorylase, indicating that, following th e endonucleolytic cleavages, fragments are normally degraded by 3 ' to 5 ' exonucleolytic trimming. Although mRNA degradation was suspected to increas e the efficiency of translational control based on several considerations, our results indicate that inhibition of mRNA degradation has no effect on t he level of repression by ThrRS. (C) 2001 Academic Press.