RT Journal Article
SR Electronic
T1 Two-step binding kinetics of tRNA<sup>Gly</sup> by the <em>glyQS</em> T-box riboswitch and its regulation by T-box structural elements
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 350413
DO 10.1101/350413
A1 Jiacheng Zhang
A1 Bhaskar Chetnani
A1 Eric D. Cormack
A1 Wei Liu
A1 Alfonso Mondragón
A1 Jingyi Fei
YR 2018
UL http://biorxiv.org/content/early/2018/06/19/350413.abstract
AB 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 T-box 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.SIGNIFICANCE Bacteria commonly use riboswitches, cis-regulatory RNA elements, to regulate the transcription or translation of the mRNAs upon sensing signals. Unlike small molecule binding riboswitches, T-box 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.AUTHOR CONTRIBUTIONS: JZ, BC, AM, and JF designed the research. JZ conducted smFRET experiments and analyzed the data. BC prepared the samples and conducted ITC experiments. EC helped with imaging and data analysis. WL and JF helped with data analysis. JZ, BC, AM, and JF wrote the manuscript; all authors approved the final manuscript.The authors declare no conflict of interest.