The interaction bacterial and phage proteins with immobilized Escherichia coli RNA polymerase.

Abstract

Abstract The interaction of Escherichia coli RNA polymerase with other proteins has been studied by affinity chromatography. Cyanogen bromide-activated agarose covalently binds RNA polymerase, and the bound protein is enzymatically active. A column containing immobilized polymerase will retain other proteins which interact with that enzyme; these proteins can be subsequently eluted and identified. The E. coli transcriptional factor σ is one such RNA polymerase-binding protein. Certain bacteriophage T4 proteins bind to polymerase-agarose, including the products of T4 maturation control genes 33 and 55. One phage T7 protein (the 0.3 protein, of unknown function) also binds to RNA polymerase. Using this convenient affinity probe, which provides a one-step analysis of crude cell extracts, I have screened related phage systems for RNA polymerase-binding proteins: T2 and T6 produce binding proteins comparable to those of T4; whereas T3 lacks a protein equivalent to the 0.3 protein of T7. Resins containing core RNA polymerase, holoenzyme (core enzyme and σ), and polymerase modified by T4 infection display different specificities for viral proteins. In particular, the T4-modified RNA polymerase column specifically retains the product of T4 DNA synthesis gene 45.