tRNA determinants for transcription antitermination of the Bacillus subtilis tyrS gene

Transcriptional regulation of the T box family of aminoacyl-tRNA synthetase and amino acid biosynthesis genes in Gram-positive bacteria is mediated by a conserved transcription antitermination system, in which readthrough of a termination site in the leader region of the mRNA is directed by a specif ic interaction with the cognate uncharged tRNA. The specificity of this int eraction is determined in part by pairing of the anticodon of the tRNA with a "specifier sequence" in the reader, a codon representing the appropriate amino acid, as well as by pairing of the acceptor end of the tRNA with an unpaired region of the antiterminator. Previous studies have indicated that although these interactions are necessary for antitermination, they are un likely to be sufficient. In the current study, the effect of multiple mutat ions in tRNA(Tyr) on readthrough of the tyrS leader region terminator, inde pendent of other tRNA functions, was assessed using a system for in vivo ex pression of pools of tRNA variants; this system may be generally useful for in vivo expression of RNAs with defined end points. Although alterations i n helical regions of tRNA(Tyr) that did not perturb base pairing were gener ally permitted, substitutions affecting conserved features of tRNAs were no t. The long variable arm of tRNA(Tyr) could be replaced by either a short v ariable arm or a long insertion of a stable stem-loop structure. These resu lts indicate that the tRNA-leader RNA interaction is highly constrained, an d is likely to involve recognition of the overall tertiary structure of the tRNA.