Single Molecule Approaches to Understanding Protein-Nucleic Acid Binding and the RNA polymerase II Transcription Reaction

The goal of this line of research is to investigate the assembly mechanism, dynamics, and heterogeneity of human transcription factor/DNA complexes, and how these parameters contribute to transcriptional control. Single-molecule studies have emerged as essential contributors to understanding the dynamic behavior, conformational states, and heterogeneity of biological complexes, thus allowing unprecedented insight into their function. Single-molecule experiments complement the knowledge gained from ensemble biochemical experiments by allowing the observation of sub-populations of molecules that exist in distinct states and also the measurement of dynamic behavior in individual molecules, which are obscured by averaging across all the molecules present in an ensemble. We are leveraging the unique abilities of the techniques of single-molecule fluorescence co-localization and single-molecule fluorescence resonance energy transfer (smFRET), which together can provide unprecedented insight into the biological function of nucleoprotein complexes. We are using these techniques to investigate the assembly and activity of complexes containing promoter DNA, transcriptional regulatory proteins, general transcription factors, and RNA polymerase II.

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