New FRET Methods for Studying Processing of Nucleic Acids by Protein Machines

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Single-molecule FRET microscopy is a versatile fluorescence technique that serves as a molecular ruler reporting on nanoscale distances and distances changes within biomolecules. During the past 15 years, studies based on single-molecule FRET have uncovered substantial information about the dynamics of nucleic acids (DNA and RNA), as well as the mechanisms of nucleic acid processing by a large variety of protein machines. Such studies are often based on direct, real-time observation of the dynamics of surface-immobilized individual molecules of nucleic acids or their complexes with proteins, and offer first views of intermediates undetectable by more conventional structural and biochemical methods.However, many single-molecule FRET techniques are often limited to a single observable, namely the FRET efficiency between the donor and acceptor probes; as a result, one may need considerable information about a system to interpret the FRET signal appropriately. In my talk, I will describe how the coupling of the FRET signal with additional fluorescence observables at the single-molecule level can be used to explore transient reaction intermediates and reaction paths. Specifically, I will discuss combinations of single-molecule FRET with alternating-laser excitation (ALEX), tethered fluorophore motion (TFM) and protein-induced fluorescence enhancement (PIFE). I will also describe examples of applications of such FRET-based techniques in mechanistic studies of gene transcription and site-specific DNA recombination. Prospects of extending FRET and its combinations in vivo will also be discussed.