The analysis of TyrS sequence revealed the typical HIGH and KMSKS domains of class I aminoacyl tRNA synthetases, being the HIGH motif perfectly conserved, and the KMSKS motif is represented by the KFGKT sequence, as in E. coli [23], Bacillus subtilis [24], and E. faecalis [14]. Mapping of the transcriptional selleckchem start site revealed a long untranslated HDAC cancer leader region of 322 bp with a highly conserved set of primary-sequence and secondary structure elements. These elements include three stem-loop structures, a highly

conserved 14-bp sequence designated the T box, and a factor-independent transcriptional terminator (Figure 3). These features are also present in other genes of gram positive bacteria, mainly genes encoding aminoacyl-tRNA synthetases, but also amino acid biosynthetic genes and transporters [25–27]. Several studies have revealed a crucial role for conserved leader region Selleck Wnt inhibitor motifs in regulation of gene expression at the level of premature termination of transcription [28]. In order to test whether this mechanism regulates the tyrS gene of E. durans TDC cluster, the

levels of mRNA were quantified using specific primers for the leader and coding region of tyrS. When E. durans was starved for tyrosine, the predominant transcript was a 1.6 kb mRNA fragment, which is the expected size for full-length mRNA (mRNA-C). Interestingly, when tyrosine was present in excess, full-length mRNA was dramatically depleted, whereas the truncated mRNA-L species kept almost constant. Thus, tyrosine had no effect on the total number of mRNA-L molecules

but caused a stoichiometric replacement of full-length mRNA by truncated RNA molecules. These data are consistent with the idea that tyrosine controls tyrS expression by promoting the Phosphoglycerate kinase premature termination of transcription rather than by inhibiting the initiation of transcription. Experiments involving transcriptional fusions of the tyrS promoter with ß-galactosidase provided evidence for this mechanism. We showed that deletion of the T box-Terminator domain of the leader region originates a complete lost of regulation by tyrosine. Early termination at pH 4.9 in presence of tyrosine observed in vivo in the leader tyrS mRNA (which shows that this sequence promotes terminator formation specifically in presence of tyrosine) was not observed for the PtyrS Δ promoter. This effect can be expected because the T box sequence is present in a side bulge of the antiterminator overlapping the terminator-antiterminator structures. In addition to the tyrosine regulation, transcription of tyrS is under strict pH control in E. durans, being expressed mostly at acidic growth conditions. The aminoacyl-tRNA synthetases catalyze the covalent attachment of amino acids to their cognate tRNAs.