A central concern of the present post-genomic era of biology is understanding the chemical and physical mechanisms by which gene expression is regulated. Appropriate activation and repression of particular genes is necessary for maintaining normal cell function and is required for executing the programs of cell differentiation that are essential to the development of multicellular organisms. Collectively, gene regulatory systems are the """"""""brain"""""""" of the cell that allows it to respond appropriately to environmental stimuli. Many cancers and other diseases result from deranged gene regulation. In this research project, we have developed and begun applying an entirely new approach to studying the molecular mechanisms of transcription and transcription regulation in vitro. Instead of studying populations of molecules, we directly visualize the RNA polymerase and regulatory proteins on an isolated single DNA molecule, following the progression of the molecular machinery through its different states in real time while simultaneously observing the extent of transcriptional activation. Such direct visualization is made possible by a multi-wavelength single-molecule fluorescence approach we call CoSMoS (co localization single- molecule spectroscopy). In this application, we propose applying the CoSMoS approach to elucidating the dynamic mechanisms of selected processes involved in regulation of transcription initiation and elongation in vitro using purified Escherichia coli proteins in reconstituted transcription reactions. Our goals are: 1) Test the hypothesis that RNAP holoenzyme molecules reach promoters through a bind-and-slide mechanism;2) Define the dynamic mechanisms by which secondary channel factors act alone and together to regulate transcription initiation and elongation;and 3) Define the dynamic mechanism by which the presence of the 70 and NusA components of the transcription apparatus is remodeled in the transition between transcription initiation and elongation.
The proposed research will elucidate basic mechanisms of transcription regulation, which in the long term will improve public health by improving our understanding of human biology. In addition, the proposed research will help define the molecular bases for regulatory mechanisms that affect virulence and environmental dissemination of human pathogens. This basic knowledge is expected to aid in the scientific research aimed at development of agents to combat infectious disease.
|Hoskins, Aaron A; Rodgers, Margaret L; Friedman, Larry J et al. (2016) Single molecule analysis reveals reversible and irreversible steps during spliceosome activation. Elife 5:|
|Harden, Timothy T; Wells, Christopher D; Friedman, Larry J et al. (2016) Bacterial RNA polymerase can retain Ïƒ70 throughout transcription. Proc Natl Acad Sci U S A 113:602-7|
|Friedman, Larry J; Gelles, Jeff (2015) Multi-wavelength single-molecule fluorescence analysis of transcription mechanisms. Methods 86:27-36|
|Ticau, Simina; Friedman, Larry J; Ivica, Nikola A et al. (2015) Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading. Cell 161:513-25|
|Yang, Boqian; Pu, Mingming; Khan, Hanif M et al. (2015) Quantifying transient interactions between Bacillus phosphatidylinositol-specific phospholipase-C and phosphatidylcholine-rich vesicles. J Am Chem Soc 137:14-7|
|Larson, Joshua; Kirk, Matt; Drier, Eric A et al. (2014) Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope. Nat Protoc 9:2317-28|
|Wang, Tracy Y; Friedman, Larry J; Gelles, Jeff et al. (2014) The covalent trimethoprim chemical tag facilitates single molecule imaging with organic fluorophores. Biophys J 106:272-8|
|Anderson, Eric G; Hoskins, Aaron A (2014) Single molecule approaches for studying spliceosome assembly and catalysis. Methods Mol Biol 1126:217-41|
|Paramanathan, Thayaparan; Reeves, Daniel; Friedman, Larry J et al. (2014) A general mechanism for competitor-induced dissociation of molecular complexes. Nat Commun 5:5207|
|Crawford, Daniel J; Hoskins, Aaron A; Friedman, Larry J et al. (2013) Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5' splice site and branch site. Proc Natl Acad Sci U S A 110:6783-8|
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