Single-molecule folding studies and genetic regulation of a S-adenosylmethionine (SAM) riboswitch / Études de la régulation génétique et du repliement du ribogétulateur SAM

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Riboswitches are structured motifs that usually reside in the non-coding region of mRNAs, where they bind metabolites and control gene expression. Unlike many other genetic control systems, riboswitches do not directly require metabolite-binding proteins to act as sensors, and thus provide a direct link between the genetic information that is encoded by an mRNA and its cellular environment. This research program will first aim to characterize the importance of residues of the S-adenosylmethionine (SAM) aptamer that are required to attain the native structure using a variety of biochemical and biophysical techniques, such as FRET (Fluorescence Resonance Energy Transfer). FRET provides a unique way of determining angles between subtended different arms in a junction and the identification of stacking partners. Key mutants will be engineered to study how riboswitch folding domains are used in the ligand recognition mechanism. The second goal will be the characterization of a recently uncovered folding transition, the ligand-dependent helical twist. The third goal of the proposed research will consist in the characterization of the riboswitch conformational changes in real time, which is only attainable using single-molecule approaches. The single-molecule FRET (sm-FRET) technique is unique in its ability to monitor dynamic structural changes of RNA molecules in real time, which provides a complete view of the folding pathway of riboswitches by revealing discrete intermediates together with their associated folding rate(s). Such measurements will be obtained using a state-of-the-art single-molecule total-internal reflection (TIR) setup coupled to an electron-multiplying CCD camera allowing observation time in the range of the low-millisecond timescale. Clearly, we expect to characterize the entire folding pathway of the riboswitch and to obtain a more complete understanding by which ligand binding to the riboswitch is harnessed to ensure a timely and efficient genetic regulation. The characterization of the SAM riboswitch will provide new insights on RNA folding mechanisms, and how it is involved in the ligand binding specificity that is crucial for the control of genetic expression. **