The overall goal is to provide a structural basis for understanding the mechanisms of initiation and elongation by DNA and RNA polymerases, their transitions from initiation to elongation phases, the regulation of polymerases by factors and the mechanisms by which these polymerases assure that the correct nucleotide is inserted. This objective will be achieved by determining the crystal structures of polymerases complexed with functionally associated proteins and bound to appropriate DNA or RNA substrates, as well as by appropriate biochemical experiments. Further, we aim to establish the structures of component assemblies of the replisome. These include a replication fork complex between DNA polymerase III, tau and a forked-DNA substrate, as well as the structure of the primasome that includes Thermus aquaticus DnaB helicase complexed with the DnaG primase bound to their DNA substrate. Additionally, the structure of the DnaB helicase bound to its DNA and nucleotide substrates as well as the helicase binding domain of DnaG and helicase loader protein. The structural basis of the initiation of DNA synthesis by the protein primed phi29 DNA polymerase and the mechanism of its transition to the elongation phase will be determined through a series of structures of intermediate states in this transition. The regulation of transcription initiation by the multi-subunit RNA polymerase from E. coli will be examined from its structures complexed with the gal and lac operon promoter DNAs and the catabolite gene activator protein regulator transcription factor. Bacterial replicating DNA polymerases as well as the transcribing RNA polymerases are targets of antibacterial antibiotics whose improvement may be aided the structures determined. As the phi29 DNA polymerase is homologous to those of several human pathogenic viruses, knowledge of its structure may yield insights into designing inhibitors of these pathogenic polymerases.
We are pursuing an understanding of the structural bases for the mechanisms by which the proteins in the replisome complex are able to copy the DNA genome into DNA and the mechanisms by which RNA polymerase is able to copy DNA into RNA as well as how transcription factors are able to regulate this process. Since the bacterial DNA polymerase and RNA polymerase differ from their eukaryotic counterparts, they are the targets of antibiotics whose improvement might be facilitated by these structures.
Showing the most recent 10 out of 38 publications
