Identification of tRNA elements important for antitermination in the T box regulatory system.

Expression of many amino acid-related genes in Gram-positive bacteria is controlled by the T box regulatory mechanism. The 5’ untranslated region of the nascent RNA (the “leader RNA”) of genes in the T box family senses the tRNA charging ratio in the cell through direct interaction with its cognate tRNA. This interaction regulates the expression of the downstream genes by determining whether the leader RNA folds into mutually exclusive terminator or antiterminator structures. Three residues within the leader RNA, termed the Specifier Sequence, resemble a codon that corresponds to the amino acid identity of the downstream genes. Both charged and uncharged tRNAs interact with the Specifier Sequence by codon-anticodon pairing, but only the free acceptor end of uncharged tRNA can form a second interaction with residues in a bulge domain of the antiterminator of the leader RNA; this interaction stabilizes the antiterminator and allows synthesis of the full-length transcript.

Previous mutational analysis revealed that pairings between the Specifier Sequence and the anticodon, and between residues in the antiterminator bulge of the leader RNA and the acceptor end of the tRNA, are necessary but not sufficient for efficient antitermination. The goal of this study is to identify tRNA residues outside of these known positions that contribute to specific interactions with the T box leader RNA. We used the Bacillus subtilis glyQS gene, which encodes glycyl-tRNA synthetase, to characterize such elements in vitro. The Specifier Sequence-anticodon pairing mimics codon-anticodon pairing; therefore, tRNA mutations that result in misreading during translation may have an effect on antitermination. We selected tRNA mutations based on conservation and suppressor tRNA studies, and introduced them into tRNA^Gly to determine their effects on glyQS antitermination and binding. The results indicate that while some mutations that cause miscoding also affect antitermination, others are tolerated in antitermination. This part of the work provides new insight into the tRNA recognition requirements in antitermination relative to that in translation, and reveals different evolutionary constraints of these processes.

We also used the Clostridium acetobutylicum alaS gene, which encodes alanyl-tRNA synthetase, to show that the cognate tRNA^Ala directs efficient alaS antitermination, but the heterologous B. subtilis tRNA^Ala (with the same anticodon and acceptor end sequences) does not. Base substitutions at positions that show sequence variations between these two tRNAs reveal complex intramolecular relationships. These experiments provide the first information about fine-tuning of tRNA to form specific interactions with its cognate leader RNA. The results imply that the T box leader RNA has coevolved with its cognate tRNA in each organism to achieve specificity and efficiency during antitermination.

Because T box riboswitches are found in pathogenic Gram-positive bacteria and regulate several essential amino acid-related genes, this system has become a novel target for antibiotic design. Previous studies have identified lead compounds that bind to the antiterminator model RNA with affinity and specificity. Here, we used in vitro and in vivo assays to test the effect of these compounds on antitermination.

Overall, the roles of T box riboswitches in tRNA evolution and clinical application are revealed.