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 goal of this proposal is to build research capacity in the state of Kentucky by establishing a research program that is competitive at the RO1 level of federal funding. The project is designed to understand mechanisms of transcription elongation control and their evolution. We propose to characterize a unique mechanism of transcription elongation control originally discovered in the bacteriophage HK022. HK022 is a temperate phage of the lambda family that infects Escherichia coli. Most members of the lambda phage family use phage encoded proteins to promote gene expression by suppressing transcription termination. HK022 has dispensed with the requirement of an antitermination protein but relies on the direct interaction of sites in the nascent RNA transcript with the host RNA polymerase. These sites, called put, are located in each of the two early operons and are comprised of two stem and loops separated by an unpaired base. The structure of the sites i s important for their function. Mutations that disrupt base pairing reduce antitermination and secondary mutations that reestablish base pairing restore terminator read through. Host mutants that prevent antitermination occur exclusively in a highly conserved zinc-finger domain located at the amino terminus of the beta prime subunit of RNA polymerase. These and other results suggest that the beta prime zinc finger recognizes put RNA and that this domain has a general role in transcription termination. By comparing the gene expression profiles of wild type E. coli with cells that carry mutations in the zinc finger, we hope to identify cellular targets of this highly conserved domain. To increase our understanding of RNA-mediated antitermination and the importance of specific RNA structural elements, we will identify and characterize additional examples of antiterminator RNAs.
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