T box riboswitches are cis-acting RNA elements that bind to tRNA and sense its aminoacylation state to influence gene expression. Here, we present the 3.2 Å resolution X-ray crystal structures of the T box Stem I-tRNA complex and tRNA, in isolation. T box Stem I forms an arched conformation with extensive intermolecular contacts to two key points of tRNA, the anticodon and D/T-loops. Free and complexed tRNA exist in significantly different conformations, with the contacts stabilizing flexible D/T-loops and a rearrangement of the D-loop. Using a designed T box RNA/tRNA system, we demonstrate that the T box riboswitch monitors the length and orientation of two essential contacts. Length or orientation mismatches engineered into the T box riboswitch and tRNA disrupt the complex, whereas simultaneous insertion of full helical turns realigns the interfaces and restores interaction between artificially elongated T box riboswitch and tRNA molecules.
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