This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The transcription of DNA into RNA is a central process in the transfer of information required for protein synthesis. The study of transcription in prokaryotes provides a simple model for understanding transcription events in all living systems. The goal of this work is to characterize the structural factors involved in transcription initiation at Class I catabolite activator protein (CAP)-dependent promoters in bacteria. To this end, a crystal structure containing CAP, DNA and the C-Terminal Domain of the ?? subunit of RNA polymerase (??-CTD), which has been recently solved in this lab, is expanded to include the C-terminus of the ?? subunit of RNA polymerase (?? Region 4 or ??-R4). The ??-R4 of RNA polymerase is thought to make specific contacts with ??-CTD and DNA in transcription initiation. Thus, a complex of CAP, DNA, ??-CTD, and ??-R4 has been crystallized. Data has been collected on NSLS X25 to 3.5 ?? resolution, but we have not yet been able to complete structure determination.We now have crystals that we believe consist of a subassembly of the CASD complex with alphaCTD, sigma region 4, and DNA (ASD). Several test images collected at the Cornell synchrotron indicate diffraction beyond 3 A, and lattice dimensions consistent with the proposed contents. If these crystals are indeed an ASD complex, determining this structure will be provide a very satisfactory means to complete several of the specific aims of this NIH-funded project.
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