Determinants for DNA Binding by a Prokaryotic Zinc-Finger Transcription Factor

Virginia Commonwealth University, Richmond VA

Investigators

Abstract

Abstract Zinc-binding proteins play a major role in nucleic acid metabolism and in the regulation of gene expression. Proteins in the bacteriophage P2 Ogr family comprise a unique group of prokaryotic zinc-finger transcription factors, which bind to an unusual DNA target site upstream of the promoters they activate. This project employs genetic and biochemical approaches to investigate the molecular basis of DNA recognition by the P2 Ogr protein and the related NucC protein encoded by a cryptic prophage in Serratia marcescens. NucC, which has higher activity in vivo and in vitro, will be used for most of the analysis. Determinants for sequence-specific DNA recognition by NucC will be probed by chemical reactivity, SELEX and crosslinking experiments. A combined approach of alanine scanning mutagenesis and isolation of specific classes of Ogr and NucC mutants will be used to define residues involved in DNA binding, transcription activation, and protein:protein interactions. Since these phage-encoded activators bear no apparent sequence or structural similarity to other known classes of prokaryotic transcription factors, elucidation of their mechanism of action will provide new insights into positive control as well as a novel mode of DNA recognition. When a virus infects a cell, the gene expression machinery of the host cell is altered by the virus and viral genes are turned on in a strictly controlled order. Dr. Christie's laboratory uses infection of a bacterial cell by the bacterial virus P2 as a model system for studying the activation of mRNA synthesis for specific genes by the interaction of viral proteins with the host transcriptional machinery. These studies are designed to provide new insights into protein:DNA interactions that play critical roles in the control of gene expression. Elucidation of molecular details underlying transcriptional regulation of gene expression is central to understanding not only viral infection but the normal control of cell growth and differentiation