The long term goal of this project is to understand the mechanism of transcription and its regulation. Determining the three-dimensional structures of RNA polymerase, the enzyme responsible for RNA synthesis, is an essential step. This is best accomplished with the highly characterize E. coli RNA polymerase. Progress on structure determination of cellular RNA polymerases has been largely due to electron microscopy and image processing of two-dimensional crystals. Previously, low resolution structures of E. coli RNA polymerase (core and holoenzyme), and yeast RNA polymerase II, were determined from crystals preserved by the method of negative staining. For each of these structures, the locations of the varied subunits and of important function sites are unknown. Here, structural studies are proposed to use the method of cryoelectron microscopy and image processing, to: 1) Determine a more detailed structure of E. coli core RNA polymerase by cryoelectron microscopy of helical crystals preserved in a frozen-hydrated state in the absence of strain. 2) Locate specific sites on the RNA polymerase by cryoelectron microscopy of site-specifically labeled enzymes. 3) Locate subunit domains within the RNA polymerase by cryoelectron microscopy of mutant enzymes harboring large insertions or deletions at known sites. 4) Locate the subunits within the RNA polymerase structure by electron microscopy. These studies will provide the most detailed view yet of a cellular RNA polymerase and provide the basic structural parameters required for the modeling and design of future experiments to elucidate the process of transcription, which is fundamental to all life.
| Opalka, Natacha; Chlenov, Mark; Chacon, Pablo et al. (2003) Structure and function of the transcription elongation factor GreB bound to bacterial RNA polymerase. Cell 114:335-45 |
| Darst, Seth A; Opalka, Natacha; Chacon, Pablo et al. (2002) Conformational flexibility of bacterial RNA polymerase. Proc Natl Acad Sci U S A 99:4296-301 |
| Opalka, N; Mooney, R A; Richter, C et al. (2000) Direct localization of a beta-subunit domain on the three-dimensional structure of Escherichia coli RNA polymerase. Proc Natl Acad Sci U S A 97:617-22 |
