Dr. Landick has submitted a proposal for continuation of his work on transcription pausing and termination of the E. coli RNA polymerase. The main topic is the pause site in the his leader. During the past project period he has made impressive progress as evidenced in the progress report and the large number of publications (19 in peer-reviewed journals, 3 reviews, and 2 submitted). Substantial progress has been made in three areas: mapping of functional domains on the subunits involved in pausing and termination, elucidated the architecture of the transcription complex and changes in this during pausing, and further definition of the his pause signal. This large body of work has made important conceptual contributions to our understanding of the transcription process. For example, the cross linking of the pausing hairpin to a specific region of the subunit and the simultaneous apparent change in the RNA polymerase conformation lends credence to the model that pausing is induced, at least in part, by such interactions. The work of Dr. Landick and his coworkers and collaborators has also generated a novel important method by which the transcription of individual RNA polymerase molecules can be monitored. Thus it is now possible to determine the components of the average results obtained from traditional molecular biology experiments. For the next project period Dr. Landick has proposed a series of logical extensions of this work. Work on the architecture will be expanded to mapping of the 3' end of the nascent transcript and the role of the pausing hairpin will be further investigated. Furthermore, it will be attempted to better map position and conformation of RNA polymerase during its translocation on the DNA template and pausing. It is hoped that this will help distinguish between inch-worm and sliding models. A very important task for the next period is to test if the NTP concentration has any effect on the events during pausing and termination of transcription. Previous experiments have all been done with a very low and unphysiological GTP concentration (5muM) in order to slow the reaction. Transcription experiments at higher GTP concentration will be monitored with video microscopy of individual polymerases as well as stop-flow kinetics. Furthermore, the role of transcription factors, such as NusA and NusG will be assessed, since these factors have been omitted from earlier experiments. Genetics will be employed to refine the definition of the structural elements of the RNA polymerase that play a functional role in pausing and termination and it will be attempted to assess possible roles of pausing in the folding of the nascent RNA transcript. Finally, genetics will also be employed to assess the in vivo role of pausing for the attenuation process in the his leader.
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