THE ESCHERICHIA-COLI THREONYL-TRANSFER-RNA SYNTHETASE GENE CONTAINS ASPLIT RIBOSOMAL-BINDING SITE INTERRUPTED BY A HAIRPIN STRUCTURE THAT IS ESSENTIAL FOR AUTOREGULATION

The expression of the gene encoding Escherichia coil threonyl-tRNA syn thetase (ThrRS) is negatively autoregulated at the translational level . ThrRS binds to its own mRNA leader, which consists of four structura l and functional domains: the Shine-Dalgarno (SD) sequence and the ini tiation codon region (domain 1); two upstream hairpins (domains 2 and 4) connected by a single-stranded region (domain 3). Using a combinati on of in vivo and in Vitro approaches, we show here that the ribosome binds to thrS mRNA at two non-contiguous sites: region -12 to +16 comp rising the SD sequence and the AUG codon and, unexpectedly, an upstrea m single-stranded sequence in domain 3. These two regions are brought into close proximity by a 38-nucleotide-long hairpin structure (domain 2). This domain, although adjacent to the 5' edge of the SD sequence, does not inhibit ribosome binding as long as the single-stranded regi on of domain 3 is present. A stretch of unpaired nucleotides in domain 3, but not a specific sequence, is required for efficient translation . As the repressor and the ribosome bind to interspersed domains, the competition between ThrRS and ribosome for thrS mRNA binding can be ex plained by steric hindrance.