A DNA-binding protein with specificity for pur genes in Escherichia coli

Abstract

A DNA-binding protein was isolated from Escherichia coli using a procedure designed for selective enrichment of regulatory proteins. In this procedure, a multicopy bacterial plasmid carrying a purE operon was used to sequester and mobilize the putative regulatory protein for pur genes. Purification of the protein from a plasmid-enriched lysate was obtained by precipitation with polyethylene glycol, elution from the precipitate with high salt and fractionation by phosphocellulose, and AMP affinity chromatography. Analysis of DNA binding by nitrocellulose filter assays showed that binding of the protein required the presence of pur genes and was either ATP dependent for some genes (purE, purA) or GTP dependent for others (purF, purI). Plasmid DNA carrying the guaAB operon did not bind the protein.