Structures of DNA and RNA Polymerases and Functional Complexes

Yale University, New Haven CT

Investigators

Linked publications & trials

Abstract

[unreadable] DESCRIPTION (provided by applicant): 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. We aim to establish the structures of component assemblies of the replisome. These include a replication fork complex with 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 that of the clamp loading protein bound to its DNA substrate, sliding clamp and single-stranded DNA binding protein will be established. The structural basis of the initiation of DNA synthesis by the protein primed <J) 29 DNA polymerase and the mechanism of transition to the elongation phase will be determined through a series of structures of intermediate states in this transition. To understand the mechanism of its transition from the initiation phase of RNA synthesis to the elongation phase, the structures of T7 RNA polymerase complexed with promoter DNA substrates and RNA transcripts of lengths 6 to 10 nucleotides will be determined. The regulation of transcription initiation by multi-subunit RNA polymerases from E. coli and M. jannaschii will be examined from their structures complexed with their respective promoter DNAs and the appropriate regulator transcription factors. To establish how the accurate addition of CCA to the immature end of tRNA is accomplished in the absence of a nucleic acid template by the class II enzymes, the structures of its complexes with tRNA or tRNA mimics missing the 3' terminal A, CA or CCA and a non-reactive analogue of the nucleoside triphosphate will be determined. 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 $29 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. [unreadable] [unreadable] [unreadable]