T-box riboswitches are cis-regulatory RNA elements that regulate the expression of proteins involved in amino acid biosynthesis and transport by binding to specific tRNAs and sensing their aminoacylation state. While the T-box modular structural elements that recognize different parts of a tRNA have been identified, the kinetic trajectory describing how these interactions are established temporally remains unclear. Using smFRET, we demonstrate that tRNA binds to the riboswitch in two steps, first anticodon recognition followed by the sensing of the 3’ NCCA end, with the second step accompanied by a T-box riboswitch conformational change. Studies on site-specific mutants highlight that specific T-box structural elements drive the two-step binding process in a modular fashion. Our results set up a kinetic framework describing tRNA binding by T-box riboswitches, and suggest such binding mechanism is kinetically beneficial for efficient, co-transcriptional recognition of the cognate tRNA ligand.

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bound form of the dye is enhanced 1100-fold. The crystal structure of the RNA-dye complex revealed a three-tiered G-quadruplex RNA structure, with the dye binding to one face of the G-quadruplex. Moreover, the folding of the aptamer is strongly coupled to the binding of the dye, thus making the dye fluorescence a direct reporter of the folding state of the G-quadruplex RNA. Previous studies by our lab of a DNA G-quadruplex, revealed that it changes conformation and eventually unfolds under high pressure. We find that pressure modifies the folding of RNA Mango-dye complex as well, and appears to significantly slow the folding reaction. In addition, it is known that potassium ions play a crucial role in stabilizing G-quadruplexes. High pressure fluorescence experiments on RNA Mango at different salt concentrations will be presented as well. These results help to better understand the folding mechanism of RNA G-quadruplexes.

SIGNIFICANCEBacteria commonly use riboswitches, cis-regulatory RNA elements, to regulate the transcription or translation of the mRNAs upon sensing signals. Unlike small molecule binding riboswitches, Tbox riboswitches bind tRNA and sense their aminoacylated state. T-box modular structural elements that recognize different parts of a tRNA have been identified, however, how each of these interactions is established temporally during tRNA binding remains unclear. Our study reveals that tRNA binds to the riboswitch in a two-step mechanism, with anticodon recognition first, followed by binding to the NCCA sequence at the 3' end of the tRNA with concomitant conformational changes in the T-box. Our results also highlight the importance of the modular structural elements of the T-box in each of the binding steps.ABSTRACT T-box riboswitches are cis-regulatory RNA elements that regulate mRNAs encoding for aminoacyl tRNA synthetases or proteins involved in amino acid biosynthesis and transport. Rather than using small molecules as their ligands, as do most riboswitches, T-box riboswitches uniquely bind tRNA and sense their aminoacylated state. Whereas the anticodon and elbow regions of the tRNA interact with Stem I, located in the 5' portion of the T-box, sensing of the aminoacylation state involves direct binding of the NCCA sequence at the tRNA 3' end to the anti-terminator sequence located in the 3' portion of the T-box. However, the kinetic trajectory that describes how each of these interactions are established temporally during tRNA binding remains unclear. Using singlemolecule fluorescence resonance energy transfer (smFRET), we demonstrate that tRNA binds to the riboswitch in a two-step process, first with anticodon recognition followed by NCCA binding, with the second step accompanied by an inward motion of the 3' portion of the T-box riboswitch relative to Stem I. By using site-specific mutants, we further show that the T-loop region of the Tbox significantly contributes to the first binding step, and that the K-turn region of the T-box influences both binding steps, but with a more dramatic effect on the second binding step. Our results set up a kinetic framework describing tRNA binding by T-box riboswitches and highlight the important roles of several T-box structural elements in regulating each binding step.