This research program deals with mechanisms of transcription and transcriptional regulation. One major project area focusses on the mechanism for coupling the expression of certain viral genes to DNA replication during the development of the bacterial virus T4, a process that is now known to involve transcriptional enhancer mechanisms. The principal research topics are: 1) the role of DNA connectivity in the enhancement (i.e. whether enhancing interactions occur in a three-dimensional space or whether they require tracking along DNA); 2) the effects of assembling replication proteins into replisome on their ability to enhance transcription and the effects of ongoing replication on enhancement; 3) figuring out how transcriptional enhancement works at the molecular level by mapping protein-protein and protein-DNA interactions at the promoter and at the enhancer. These four broad topics will be pursued by various in vitro methods, including transcription kinetics, various footprinting techniques, photocrosslinking of protein to DNA, other crosslinking methods and other methods of analyzing the binding of proteins to DNA in assemblies of moderate complexity. A fourth topic involves certain in vivo correlates of the preceding analysis. The second project area consists of several research topics involving transcription by RNA polymerase III. It is now clear that only one transcription factor of the yeast RNA polymerase III, TFIIIB, is essential for positioning the enzyme at the start site for transcription. One research topic analyzes this minimal transcription initiation system in kinetic and structural terms, using normal and mutant promoters. A second research topic involves the use of photochemical methods, including the use of a newly developed photocrosslinking reagent, to analyze the structure of transcription complexes and to map protein-protein as well as protein-DNA interactions along an entire transcription unit. A third research topic involves kinetic and structural analysis of RNA chain elongation and termination by RNA polymerase III. The connecting thread of these two lines of research is their common focus on a molecular understanding of transcription and its regulation and on processes such as promoter finding, promoter opening, modulations of promoter strength by protein-protein and protein-DNA interactions, RNA chain elongation and termination that are common to transcription in all organisms and all types of cells.
| Kassavetis, George A; Prakash, Prachee; Shim, Eunjung (2010) The C53/C37 subcomplex of RNA polymerase III lies near the active site and participates in promoter opening. J Biol Chem 285:2695-706 |
