The long-range goal of this project is to define the interactions in the transcription complex that cause pausing and termination by RNA polymerase. RNA secondary structures are important regulatory signals in prokaryotes, where transcriptional pausing and termination are major components of genetic regulatory mechanisms. Pausing and premature termination also affect gene expression in mammalian cells and viruses. Changes in pausing or termination influence expression of genes that are involved in development of cancer and in the growth of the AIDS virus, HIV-1. the underlying mechanisms for pausing and termination in mammalian cells are unknown and difficult to study. Prokaryotic systems are more amendable to investigation. This project will further understanding of transcriptional pausing and termination in E. coli, which is an excellent model system for studying the fundamental interactions in the transcription complex. Work on the model system also will provide techniques and concepts that will facilitate work on eukaryotic transcription complexes. A combined biochemical and genetic approach will be used to dissect the interactions in the bacterial transcription complex. Systematic variation of pause and termination RNA hairpin sequences and structures will define the features that influence elongation by RNA polymerase. Defined transcription complexes will be isolated and their structures probed using a variety of agents that can reveal the configuration of RNA and DNA strands within them. These biochemical studies are complemented by a genetic analysis of RNA polymerase. Both the beta and beta1 subunits contain regions where amino-acid changes alter recognition of the pause and termination signals. A set of mutant enzymes with changes in these different regions will be isolated and studied to reveal the potential role of each in the events that occur at the pause and termination sites. These experiments will be used to test alternative models for transcriptional termination, one that simple base-pairing equilibria account for termination and the second that termination is a multi-step process that RNA polymerase actively catalyzes.
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