Single-molecule fluorescence analysis of the temperature dependent structure and dynamics of an RNA thermometer: consequences for its molecular function

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Single molecule fluorescence spectroscopy with multiparameter detection has proven to be valuable tool for resolving static and dynamic heterogeneity of an ensemble. The main goal of this project is the investigation of the three-dimensional architecture and temporal dynamics of two RNA thermometers (full-length ROSE and fourU) using quantitative fluorescence resonance energy transfer (FRET) measurements. To exploit the full capabilities of these FRET studies, we establish tools for RNA structure prediction by comparative modelling using restraints derived from experimental data. Chemically synthesized RNAs will be used In the planned studies, which are specifically fluorescently labeled using custom designed dye linkers. The focus of this project is elucidating its Mg2+- and temperature-dependent conformational dynamics and structural transitions. All known RNA thermometers are cis-acting modulators that control translation initiation by masking the Shine Dalgarno (SD) sequence at low temperatures. Their architecture involves several stem loops and junction loops, whereby the temperature sensitive SD element is located in one helix arm. The overall thermometer structure and the function of the other helices are unclear so far, so that we want to establish a mechanistic knowledge between its 3D architecture and biological function.