The Bacillus subtilis tyrS gene, encoding tyrosyl-tRNA synthetase, is a member of the T-box family of genes, which are regulated by control of readthrough of a leader region transcriptional terminator. Readthrough is induced by interaction of the cognate uncharged tRNA with the leader; the system responds to decreased tRNA charging, caused by amino acid limitation or insufficient levels of the aminoacyl-tRNA synthetase. Recognition of the cognate tRNA is mediated by pairing of the anticodon of the tRNA with the specifier sequence of the leader, a codon specifying the appropriate amino acid; a second interaction between the acceptor end of the tRNA and an antiterminator structure is also important. Certain switches of the specifier sequence to a new codon result in a switch in the specificity of the amino acid response, while other switches do not. These effects may reflect additional sequence or structural requirements for the mRNA-tRNA interaction. This study includes investigation of the effects of a large number of specifier sequence switches in tyrS and analysis of structural differences between tRNA Tyr and tRNA species which interact inefficiently with the tyrS leader to promote antitermination.Many aminoacyl-tRNA synthetase and amino acid biosynthesis operons in gram-positive bacteria are regulated by a common transcription antitermination system (11,12,16,28). The genes in this group share a number of features in the 5Ј region in their transcripts, including a set of conserved primary sequence and structural elements located upstream of the start of the coding sequence. The mRNA leader region structural elements include three large stem-loops, designated stems I, II, and III, preceding a factor-independent transcriptional terminator. The most prominent of the sequence elements, the 14-base T box, forms a portion of an antiterminator structure by pairing with conserved sequences on the 5Ј side of the terminator. Expression of the genes in this group is therefore controlled at the level of readthrough of the leader region transcriptional terminator, by a switch between the antiterminator and terminator forms of the transcript.Expression of several genes in this family has been shown to be induced in response to limitation for the cognate amino acid and not by general amino acid starvation (5,6,18,27,29). Examination of a number of the leader regions revealed a precisely located single codon, specifying the appropriate amino acid (11); the effector signalling amino acid limitation was therefore proposed to be tRNA, with the specificity of the amino acid response dictated by codon-anticodon pairing. The induction specificity of the Bacillus subtilis tyrS gene was switched from a response to tyrosine limitation to a response to phenylalanine limitation by a single base substitution in the UAC tyrosine codon of the tyrS leader to a UUC phenylalanine codon (11). These studies indicated that the leader region codon, termed the "specifier sequence," acts as a critical determinant of the specificity of the amino a...