Revealing correlated movements in biomolecular complexes: transcription termination by Rho helicase

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Helicases are widespread molecular motor enzymes required for numerous essential nucleic-acid transactions. The hexameric helicase Rho is a transcription termination factor that binds to the nascent RNA and dissociates it from the transcription complex. It is the aim of this project to visualize the action of single Rho helicases and to dissect their working mechanism using multi-color singlemolecule spectroscopy. The single-molecule approach will enable to monitor specific structural changes under biologically relevant conditions in real time. Expanding the recently developed multicolor capability we intend (i) the quantitative determination of stoichiometries of biomolecular complexes, (ii) the determination of 3D structures based on Fluorescence Resonance Energy Transfer (FRET) between several fluorophores, and (iii) the visualization of biomolecular dynamics including relative movements of different segments within biomolecular complexes. The methodology will be evaluated using DNA junctions that are promising candidates for FRET based fluorescent sensors. Subsequently we will investigate the dynamics of Rho helicase activity using fluorescent labels on Rho, RNA and DNA. Important questions e.g. whether Rho leaves its initial binding site upon translocation can thus be answered. The vision is to address problems of increasing complexity with single- molecule techniques such as the reconstitution of the transcription machinery in vitro.