Abstract
The T box riboswitch regulates expression of amino acid-related genes in Gram-positive bacteria by monitoring the aminoacylation status of a specific tRNA, the binding of which affects the folding of the riboswitch into mutually exclusive terminator or antiterminator structures. Two main pairing interactions between the tRNA and the leader RNA have been demonstrated to be necessary, but not sufficient, for efficient antitermination. In this study, we used the Clostridium acetobutylicum alaS gene, which encodes alanyl-tRNA synthetase, to investigate the specificity of the tRNA response. We show that the homologous C. acetobutylicum tRNAAla directs antitermination of the C. acetobutylicum alaS gene in vitro, but the heterologous Bacillus subtilis tRNAAla (with the same anticodon and acceptor end) does not. Base substitutions at positions that vary between these two tRNAs revealed synergistic and antagonistic effects. Variation occurs primarily at positions that are not conserved in tRNAAla species, which indicates that these non-conserved residues contribute to optimal antitermination of the homologous alaS gene. This study suggests that elements in tRNAAla may have coevolved with the homologous alaS T box leader RNA for efficient antitermination.
Highlights
Gram-positive bacteria utilize the T box riboswitch to regulate the expression of genes that encode aminoacyl-tRNA synthetases, amino acid biosynthesis enzymes, and transporters [1,2].The leader region of most genes in the T box family contains an intrinsic transcriptional terminator that prevents transcription of the downstream gene; folding of the nascent transcript into a competing antiterminator structure, which is stabilized by binding of the cognate uncharged tRNA, allows synthesis of the full-length transcript
Stabilization of the antiterminator requires a second interaction between the free acceptor end (NCCA) of the uncharged tRNA and a complementary sequence (UGGN) embedded in the antiterminator bulge [4]; this interaction is prevented by the amino acid at the 3’ end of charged tRNA
When both substitutions were introduced (AbCdE), the RTmax was reduced from 32% to 20% while the K1/2 increased by 8.6-fold in comparison with C. acetobutylicum tRNAAla (UGC); this indicates that substitutions at the central core and T stem antagonized each other and resulted in reduced efficiency, despite the observation that each substitution alone had a positive effect
Summary
Gram-positive bacteria utilize the T box riboswitch to regulate the expression of genes that encode aminoacyl-tRNA synthetases, amino acid biosynthesis enzymes, and transporters [1,2]. Correct Specifier Sequence-anticodon and acceptor end-antiterminator bulge pairings are necessary for antitermination, tRNA elements outside of these two major sites contribute to efficient interaction with the cognate leader RNA [8,19,20]. This study investigates tRNA determinants important for efficient antitermination of the alaS gene, in comparison to previous findings for the glyQS and tyrS genes, to identify general features of tRNA recognition by T box leader RNAs of different structural classes. This work provides insight into how tRNA sequence requirements for antitermination are superimposed upon translational requirements
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