Direct Evaluation of tRNA Aminoacylation Status by the T-Box Riboswitch Using tRNA-mRNA Stacking and Steric Readout

•An efficient, broadly applicable method to obtain purified aminoacyl-tRNAs•T-box riboswitches directly sense tRNA aminoacylation status without proteins•T-boxes gauge the molecular volume of tRNA 3′ substituents to detect aminoacylation•Intermolecular coaxial stacking with uncharged tRNA stabilizes T-box antiterminator

SummaryT-boxes are gene-regulatory mRNA elements with which Gram-positive bacteria sense amino acid availability. T-boxes have two functional domains. Stem I recognizes the overall shape and anticodon of tRNA, while a 3′ domain evaluates its aminoacylation status, overcoming an otherwise stable transcriptional terminator if the bound tRNA is uncharged. Although T-boxes are believed to evaluate tRNA charge status without using any proteins, this has not been demonstrated experimentally because of the instability of aminoacyl-tRNA. Using a simple method to prepare homogeneous aminoacyl-tRNA, we show that the Bacillus subtilis glyQS T-box functions independently of any tRNA-binding protein. Comparison of aminoacyl-tRNA analogs demonstrates that the T-box detects the molecular volume of tRNA 3′-substituents. Calorimetry and fluorescence lifetime analysis of labeled RNAs shows that the tRNA acceptor end coaxially stacks on a helix in the T-box 3′ domain. This intimate intermolecular association, selective for uncharged tRNA, stabilizes the antiterminator conformation of the T-box.

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