This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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. We now have crystals of containing Bromo-uridine derivatized DNA. The Bromine atoms, used in isomorphous replacement and combined with a partial molecular replacement solution will permit confident placement of the DNA in the crystal. The inclusion of sigma region 4 this crystal complex bridges the gap in structural information between CAP ?????"""""""" DNA structures and RNA polymerase structures. Completion of this structure will provide a complete picture of bacterial transcription activation by CAP.
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