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 synthetase, is negatively autoregulated at the translational level. Regulation is due to the binding of threonyl-tRNA synthetase to its own mRNA at a site called the operator, located immediately upstream of the initiation codon. The present work investigates the relationship between regulation and mRNA degradation. We show that two regulatory mutations, which increase thrS expression, cause an increase in the steady-state mRNA concentration. Unexpectedly, however, the half-life of thrS mRNA in the derepressed mutants is equal to that of the wild-type, indicating that mRNA stability is independent of the repression level. All our results can be explained if one assumes that thrS mRNA is either fully translated or immediately degraded. The immediately degraded RNAs are never detected due to their extremely short half-lives, while the fully translated messengers share the same half-lives, irrespective of the mutations. The increase in the steady-state level of thrS mRNA in the derepressed mutants is simply explained by an increase in the population of translated molecules, i.e. those never bound by the repressor, ThrRS. Despite this peculiarity, thrS mRNA degradation seems to follow the classical degradation pathway. Its stability is increased in a strain defective for RNase E, indicating that an endonucleolytic cleavage by this enzyme is the rate-limiting process in degradation. We also observe an accumulation of small fragments corresponding to the 5' end of the message in a strain defective for polynucleotide phosphorylase, indicating that, following the endonucleolytic cleavages, fragments are normally degraded by 3' to 5' exonucleolytic trimming. Although mRNA degradation was suspected to increase the efficiency of translational control based on several considerations, our results indicate that inhibition of mRNA degradation has no effect on the level of repression by ThrRS.
Nogueira
T
T
Institut de Biologie Physico-Chimique, UPR9073 du CNRS, 13 rue Pierre et Marie Curie, Paris, 75005, France.
de Smit
M
M
Graffe
M
M
Springer
M
M
D004722:Endoribonucleases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006207:Half-Life; D008957:Models, Genetic; D009154:Mutation / Q000235:genetics; D011117:Polyribonucleotide Nucleotidyltransferase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics*; D020871:RNA Stability / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013914:Threonine-tRNA Ligase / Q000235:genetics*
D012329:RNA, Bacterial; D012333:RNA, Messenger; D011117:Polyribonucleotide Nucleotidyltransferase; D004722:Endoribonucleases; C022537:ribonuclease E; D013914:Threonine-tRNA Ligase