REGULATION OF BACILLUS SUBTILIS TRNA SYNTHETASE GENES

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The goal of this project is the analysis of the regulation of the Bacillus subtilis tyrS gene, encoding tyrosyl-tRNA synthetase. Aminoacyl tRNA synthetases are critical components of the translational machinery of all cells, catalyzing the accurate charging of tRNA with the cognate amino acid. Expression of tRNA synthetase genes responds to the cellular requirement for the synthetases. The mechanisms for regulation of tRNA synthetase genes has been partially characterized in the Cram-negative bacterium Escherichia coli, and have been found to be complex; no information is available about regulation of these genes in other systems. B. subtilis is a Gram-positive, spore-forming soil bacterium, evolutionarily distant from E. coli, but with a well-developed genetic system which greatly facilitates genetic analysis. The B. subtilis tyrS gene has been cloned, and its DNA sequence has been determined. Preliminary studies on tyrS regulation indicated that this gene is likely to be regulated by a transcription antitermination mechanism, in response to starvation for tyrosine. A potential regulatory target site was identified which is conserved in a number of Bacillus tRNA synthetase genes, as well as in the ilv-leu biosynthetic operon; deletion of this sequence results in an uninducible phenotype. The conservation of this site, in all cases upstream of a leader region transcriptional terminator, suggests the possibility that these genes are regulated by a common mechanism. The proposed transcription antitermination mechanism for tyrS regulation will be tested by transcriptional mapping in vivo and in vitro. The physiological conditions to which tyrS responds will be further explored. The cis-acting sequences important for tyrS regulation will be examined, and trans-acting factors required for antitermination will be identified by genetic and biochemical analysis. This study is expected to provide general information about transcription antitermination systems. This is the first report of an antitermination system proposed to operate on a large number of genes; analysis of the mechanisms by which this system is directed to act on specific genes in response to appropriate physiological signals will provide general insight into the generation of specificity in regulatory system.