DNA-dependent RNA polymerase is the key enzyme responsible for the biosynthesis of RNA, a process known as transcription. This process, which decodes the genetic information from DNA, is one of the most significant events in a biological system. Our research objective is to provide detailed information through systematic crystallographic studies to help understand the structure-function relationship of RNA polymerase at the atomic level. During the current grant period we have grown a number of crystals related to the bacteriophage T7 RNA polymerase. The crystal structure of the T7 atomic model for a RNA polymerase. We have also solve the structure of a chimeric T7/T3 RNA polymerase with T3 promoter specificity. A preliminary comparison of these two structures indicated that the """"""""thumb"""""""" subdomain of T7 RNA polymerase is not rigid and contains a hinge or """"""""knuckle"""""""" region at which it may bend to wrap around the template. In this renewal application, we aim to: (1) refine the crystal structure of T7 RNA polymerase to its highest possible resolution so that we may provide the most accurate structural model for T7 RNA polymerase, (2) refine the crystal structure of the chimeric T7/T3 RNA polymerase also to its highest possible resolution so that we may explain how the structural differences between T7 and T3 RNA polymerase promoter recognition domains determine their promoter specificities, (3) study other mutants that have altered promoter recognition, (4) determine the structures of selected deletion mutants to define the role of parts of the polymerase molecule, (5) prepare crystals and to solve the structure of transcriptional elongation complexes of T7 RNA polymerase with synthetic RNA-DNA bubble duplexes to observe directly the various interactions between the polymerase and the structures of subunits of E. coli RNA polymerase to pave the way for understanding the structures of multi-subunit RNA polymerase, (7) use the amino acid residues found in the DNA-RNA binding pocket of this large, multi-subunit enzyme. We believe our studies will provide new knowledge on the structural basis of transcription at the atomic level, and will form the basic for other genetic, biochemical and biophysical studies for the understanding of this important class of enzymes.
