Combined Optical Trapping and Fluorescence Resonance Energy Transfer

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DESCRIPTION (provided by applicant): Our current research focuses on RNA polymerase (RNAP). RNAP is an important target of genetic regulation, because of its role in almost all genetic pathways. RNAP frequently undergoes site-specific pausing, which affects recruitment of regulatory factors, transcription coupled repair, and termination. However, the specific structural mechanism which underlies pausing is not well understood. We can detect pausing of RNAP during elongation using optical tweezers, and by combining this with single molecule fluorescence resonance energy transfer (smFRET), we will detect structural changes in RNAP as a result of pausing.) Our specific aims are: 1) Develop a system for combining OT and smFRET measurements. We will present data showing that we have already accomplished this aim. 2) Apply this instrument to the study of RNA tertiary structure. Specifically, we plan to validate the instrument by using combined OT and FRET to study the how the tertiary contacts of the hammerhead ribozyme lead to changes in its structure and function. And, 3) Learn about the mechanism of RNAP pausing by observing the coordination of pausing with structural changes of the molecule. We will collaborate with Robert Landick to produce FRET-labeled RNA polymerase molecules suitable for FRET measurements, and use this, in conjunction with our instrument, to expand upon already existing Block-lab optical trapping techniques for studying RNAP pausing. RNAP pausing has a huge impact on regulation of RNAP transcription, and can lead to or prevent premature termination of mRNA transcription. Because of this, and because of RNAP's central role in genetic regulation, a better understanding of the mechanisms of RNAP pausing would have a significant impact on our understanding of genetic regulation as a whole, which in turn impacts almost every aspect of genetics, such as disease, drug resistance, and diversity.