STABILIZED SECONDARY STRUCTURE AT A RIBOSOMAL-BINDING SITE ENHANCES TRANSLATIONAL REPRESSION IN ESCHERICHIA-COLI

The expression of the gene encoding Escherichia coli threonyl-tRNA syn thetase is negatively autoregulated at the translational level. The ne gative feedback is due to the binding of the synthetase to an operator site on its own mRNA located upstream of the initiation codon. The pr esent work describes the characterisation of operator mutants that hav e the rare property of enhancing repression. These mutations cause (1) a low basal level of expression, (2) a temperature-dependent expressi on, and (3) an increased capacity of the synthetase to repress its own expression at low temperature. Surprisingly this enhancement of repre ssion is not explained by an increase of affinity of the mutant operat ors for the enzyme but by the formation, at low temperature, of a few supplementary base-pairs between the ribosomal binding site and a norm ally single-stranded domain of the operator. Although this additional base-pairing only slightly inhibits ribosome binding in the absence of repressor, simple thermodynamic considerations indicate that this is sufficient to increase repression. This increase is explained by the c ompetition between the ribosome and repressor for overlapping regions of the mRNA. When the ribosomal binding site is base-paired, the ribos ome cannot bind while the repressor can, giving the repressor the adva ntage in the competition. Thus, the existence of an open versus base-p aired equilibrium in a ribosomal binding site of a translational opera tor amplifies the magnitude of control. This molecular amplification d evice might be an essential component of translational control conside ring the low free repressor/ribosome ratio of the low affinity of tran slational repressors for their target operators. (C) 1995 Academic Pre ss Limited