Development of advanced single-molecule fluorescence imaging methods to visualize coupled conformational changes in transcription

Coupling between mobile elements of a biological molecular machine, as well as between different molecular machines, is key for the function of biological machinery and for the regulation of most biological processes. However, capturing the transient coupled dynamics of such protein machines is difficult to perform at the single-molecule level, mainly due to the absence of robust methodology for the direct observation of coupled molecular motions. This project focuses on developing single-molecule fluorescence methodology that utilizes multi-colour Foerster resonance energy transfer based on single-molecule spectra to capture coupled conformational transitions in protein interactions with nucleic acids. The new methods will be applied to the motions of modules of RNA polymerase (the machine responsible for copy DNA into messenger RNA) and the DNA molecules processed by RNA polymerase. This new methodology and its application to transcription initiation should provide a much more thorough understanding of fundamental steps in gene expression and offer new opportunities for the generation of new antibiotics targeting the RNA polymerase; further, the technological innovation should be applicable to other biomolecular complexes and processes, and thus should be of interest to the communities of microscopy developers and molecular biologists.