Transcriptional Attenuator in Bacillus subtilis

It has been proposed that uncharged tRNA molecules may act as positive regulatory factors to control the expression of a number of operons in Bacillus subtilis and related bacteria by interacting with leader sequences to cause antitermination. In this study we report the isolation and characterization of regulatory mutations that modify one of the tRNA molecules predicted to have such a regulatory role. Three different alleles of the B. subtilis leucine tRNAgene leuGwere found that resulted in higher expression of the ilu-leu biosynthetic operon. Each resulted in a base change in the D-loop of the leucine tRNA molecule with the anticodon 5’-GAG3’ (leucine tRNbAG). Experiments with strains that are diploid for mutant and wild-type alleles suggested that both charged and uncharged tRNA molecules may interact with leader sequences to control expression of the operon. B ACTERIA have evolved elaborate mechanisms to regulate gene expression in response to changes in physiological conditions and nutrient supply. The regulation of genes involved in amino acid biosynthesis in the gram-negative bacterium Escherichia coli has been studied in considerable detail. In recent years there has been increasing interest in the mechanisms that control the expression of corresponding genes in Bacillus subtilis, a gram-positive soil bacterium that occupies a very different ecological niche. In this report we describe mutations in a leucine tRNA gene that cause overexpression of the B. subtilis ilv-leu operon, which contains genes for branched-chain amino acid biosynthesis. Our analysis suggests that the operon is controlled by an attenuation mechanism in which a particular tRNA species helps to determine the frequency of transcriptional termination at a site in an untranslated leader sequence. The ilv-leu operon of B. subtilis contains seven genes required for the biosynthesis of leucine, isoleucine and valine (VANDEYAR 1987), preceded by a 482-bp untranslated leader sequence. Transcription of the operon is about 30-fold higher when cells are grown in limiting leucine than when they are grown in excess leucine (GRANDONI et al. 1993). Attenuation of transcripts within this leader accounts for most of the negative effect of excess leucine on expression of the operon (GRANDONI et al. 1992, 1993). GRUNDY and HENKIN (1993,1994) have proposed that a number of the aminoacyl-tRNA synthetase genes and amino acid biosynthetic operons of B. subtilis and related bacteria are regulated by a common mechanism in which uncharged tRNA molecules act as positive r e p lators by interacting directly with leader mRNA to promote transcriptional antitermination. Mutational analysis of the leader of the B. subtilis tyrosyl-tRNA synthetase Genetics 137: 627-636 (July, 1994) gene ( tyrS) led them to propose this novel model of transcriptional attenuation. This proposed mechanism differs from the extensively studied mechanisms that regulate the amino acid biosynthetic operons of E. coli. Translation of upstream open reading frames is an essential component of the E. coli attenuation mechanisms (LANDICK and YANOFSKY 1987). In the GRuNDYand HENKIN model, the mechanism by which tRNA molecules influence transcriptional termination is independent of translation. Analysis of the ilv-leu operon leader sequence suggests that it is regulated by this mechanism (GRUNDY and HENKIN 1994; GRANDONI et al. 1993). In the leader region of each of the operons that GRUNDY and HENKIN analyzed they identified a potential RNA secondary structure that could serve as a transcrip tional terminator. Upstream of the terminator is a potential stem-loop structure that contains an unpaired bulge with a triplet sequence (the specifier) that they believe is a site of interaction with the anticodons of regulatory tRNA molecules (GRUNDY and HENKIN 1993). In the t y S operon they showed that this site determines the specificity of the regulation. The tyrS leader has the tyrosine codon 5’-UAG3’ (UAC) at the specifier position. Changing the triplet to UUC, which codes for phenylalanine, altered the regulation so that transcrip tion of the operon became dependent on starvation for phenylalanine instead of tyrosine. They noted in their papers that the leucyl-tRNA synthetase ( l e d ) and iluleu operons both have the specifier triplet CUC that codes for leucine. Their model predicts that tRNA molecules that recognize CUC control expression of both of the operons. In this paper we describe our analysis of mutations within a leucine tRNhAG gene (ZeuG) of B. subtilis that cause overexpression of the ilv-leu 628 D . B. Garrity and S. A. Zahler