Transcription is the major control point in the pathway of gene expression. Defective transcription regulation underlies many disorders. Many oncoproteins, including the most common in a wide range of cancers, are transcriptional activator proteins. There is a remarkable degree of conservation of RNA polymerase structure and transcription mechanism across species. Thus, structure determination of bacterial RNA polymerase will reveal information that is directly applicable to understanding transcription in general. The long term objective of this project is to understand the physical mechanism of transcription and its regulation. To this end we propose the following: 1) Determine the structure of Escherichia coli RNA polymerase core enzyme in helical crystals by cryoelectron microscopy and image processing. A structural analysis of core enzyme, and comparison with the holoenzyme structure, will reveal the location of the 70 kDa sigma subunit and conformational changes that may result from sigma70 binding to core. This information will shed light on the mechanism of transcription initiation and will provide a more detailed structure of a multisubunit RNA polymerase than has ever been available. 2) Localize the active center and rifampicin binding site on the RNA polymerase structure through the study of site-specifically labeled polymerases. For these studies we will use undecagold, a specific cluster of 11 gold atoms only 8 Angstrom in diameter. When incorporated into a crystal, the electron-dense cluster can be localized by electron microscopy and image processing using the method of difference Fourier analysis with the unlabeled structure.
