Abstract
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.
Highlights
Riboswitches are cis-regulatory RNA elements that recognize and respond to defined external signals to affect transcription or translation of downstream messenger RNAs (Breaker, 2012; Serganov and Nudler, 2013; Sherwood and Henkin, 2016)
Bacterial T-box riboswitches represent a unique class of riboswitches that do not bind small molecule ligands, instead they recognize and bind transfer RNA (tRNA) molecules and sense directly their aminoacylation state (Zhang and Ferre-D’Amare, 2015)
Our results demonstrate that tRNA binds to the riboswitch in two steps, with its anticodon being recognized first, followed by NCCA binding accompanied with an inward motion of the 3’ region of the T-box riboswitch, including Stem III and the anti-terminator stem, relative to Stem I
Summary
Riboswitches are cis-regulatory RNA elements that recognize and respond to defined external signals to affect transcription or translation of downstream messenger RNAs (mRNAs) (Breaker, 2012; Serganov and Nudler, 2013; Sherwood and Henkin, 2016). T-box riboswitches are found in Gram-positive bacteria and are usually located in the region upstream of mRNA sequences encoding aminoacyl tRNA synthetases and proteins involved in amino acid biosynthesis and transport and participate directly in amino acid homeostasis (Zhang and Ferre-D’Amare, 2015). The distal bulge and the apical loop fold into a compact structural module of interdigitated T-loops (Chan et al, 2013; Krasilnikov and Mondragon, 2003), which interact directly with conserved unstacked nucleobases at the tRNA elbow (Grigg et al, 2013; Zhang and Ferre-D’Amare, 2013).
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