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Regulation mechanisms of TPP riboswitches

Riboswitches are non-coding elements upstream or downstream of messenger RNAs that, upon binding of a specific ligand, regulate transcription or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitch aptamers domains of thiC and thiM in E. coli regulating transcription and translation, respectively, and that of THIC in A. thaliana. For all, we ascertained an induced-fit mechanism with initial loose binding of TPP (characterized by the parameters kon and koff) subsequently transformed into tight binding after RNA conformational change (characterized by the parameters kF and kU). Using our kinITC approach, we obtained the temperature dependence of all kinetic and thermodynamic parameters for the bacterial riboswitches. The results imply a kinetic regulation, which requires that the RNA polymerase pauses after synthesis of the riboswitch aptamer. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentrations range of TPP. We verified the existence of these pauses and the prediction of the model on their duration. The A. thaliana riboswitch, on the contrary, is under classical thermodynamic control and showed considerable temperature-dependence of its kinetics of folding, which suggests that it could also act as a thermosensor. Our analysis also showed that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation

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