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 specific interaction with the cognate uncharged tRNA. The specificity of this interaction is determined in part by pairing of the anticodon of the tRNA with a “specifier sequence” in the leader, 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 unlikely to be sufficient. In the current study, the effect of multiple mutations in tRNATyr on readthrough of the tyrS leader region terminator, independent of other tRNA functions, was assessed using a system for in vivo expression of pools of tRNA variants; this system may be generally useful for in vivo expression of RNAs with defined end points. Although alterations in helical regions of tRNATyr that did not perturb base pairing were generally permitted, substitutions affecting conserved features of tRNAs were not. The long variable arm of tRNATyr could be replaced by either a short variable arm or a long insertion of a stable stem-loop structure. These results indicate that the tRNA–leader RNA interaction is highly constrained, and is likely to involve recognition of the overall tertiary structure of the tRNA.