Initiation and Elongation in T7 RNA Polymerase

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DESCRIPTION (provided by applicant): Understanding genetic regulation is key to understanding human disease and to exploiting the wealth of information arising in the post-genomic era. Transcription, the controlled copying of RNA from DNA, is perhaps the premier step at which this regulation (or misregulation, in the case of many diseases) occurs. This key cellular process is carried out by a molecular machine with complex function and underlying requirements. While the molecular basis of transcription has been the focus of extensive study for 50 years, it has been only fairly recently that we have seen the determination of a variety of high resolution crystal structures for the multisubunit bacterial and eukaryotic RNA polymerases, and exciting new structures for the single subunit phage polymerase from bacteriophage T7. The latter presents an ideal model system for the study of fundamental issues in transcription. Although structurally distinct from the multi-subunit RNA polymerases, it shares many common functional and mechanistic attributes. Key questions in this work will focus on the balance of energetics in this complex molecular machine. We will test and refine specific models to explain a large rearrangement within the protein known to be essential as the enzyme leaves the promoter recognition site and transitions to an elongation complex capable of stably transcribing thousands of bases. Classic enzymology will be combined with protein mutagenesis and the tools of biophysical chemistry to test and further refine detailed models for structure and function. These studies will provide a foundation from which to understand energetics and mechanism in the key transition from initiation to elongation. Functional homologies suggest that the underlying lessons learned will be applicable to all RNA polymerases.