tRNA-dependent conformational dynamics and folding coordinate translational regulation by a full-length T-box...

T-box riboswitches regulate gene expression by sensing tRNA aminoacylation status, but their dynamic mechanisms remain elusive. Here, we present single-molecule FRET studies of a full-length translational ileS T-box, showing that its decoding domain folds independently, and the initial tRNA anticodon binding promotes proper folding of the discriminator domain. Subsequent uncharged tRNA binding stabilizes the Antisequestrator (AntiS) conformation and GAG linker, resulting in translation initiation. Conversely, charged tRNA binds to a distinct T-box conformation, of which the linker is highly flexible and stem III is away from AntiS, rendering irreversible transition to the Sequestrator conformation if downstream sequences are transcribed, leading to translation inhibition. Collectively, both steps of tRNA binding are in a conformational selection manner, and the GAG linker, especially G96, acts as the main linchpin in tRNA 3΄-termini sensing. An elaborate model is proposed to understand how cotranscriptional folding, stepwise tRNA binding and dynamic conformational transitions coordinate T-box regulation.