Investigation of RNA-Processing Protein Dynamics with Simultaneous High-Resolution Optical Traps and Single-Molecule Fluorescence

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Title: Investigation of RNA-processing protein dynamics with simultaneous high-resolution optical traps and single-molecule fluorescenceIn this project, the PI will develop optical methods that will enable direct observation in real-time of the activity of protein machines that are involved in processing RNA. RNA is one of the essential carriers of cell information, and its production and degradation are tightly controlled by RNA protein interactions associated with protein machines. The overall goal of this project is to investigate how both the cooperation and competition between different molecular machines lead to the careful regulation of information in the cell. The techniques developed in this project will apply beyond the protein machines involved in RNA processing to include a broad assortment of complex protein systems. This project will leverage optical methods development and the strong art and science connection at Michigan State University to develop an education and outreach program centered on hands-on interactions with biophysical science at the individual cell and RNA molecule level. The PI will create a video game lab experience aimed at students and the public.This project will investigate RNA-processing protein dynamics of single proteins and larger protein machine assemblies by developing and applying high-resolution, multi-modal single molecule techniques. Specifically, the PI will investigate the yeast nuclear RNA helicase Mtr4p and the associated TRAMP protein complex. Mtr4p and TRAMP have emerged as key players in nuclear RNA surveillance and processing in part via precise modification of RNA for degradation by the exosome. How these machines coordinate or compete to achieve function is controversial in large part due to their highly dynamic, multi-degree of freedom interactions on the scale of only 5-10 nucleotides of RNA. The PI will address the fundamental questions regarding how Mtr4p and TRAMP function using single-molecule and high resolution optical tweezers and fluorescence. Advanced techniques combining high-resolution optical trapping and multi-color fluorescence will apply broadly across all biophysical molecular dynamics investigations towards the emerging need for tools to investigate protein-nucleic acid interactions beyond the individual molecule scale towards multi-protein, multi-degree of freedom complex systems.