A Salt-promoted Inhibitor of RNA Polymerase Isolated from T4 Phage-infected E. Coli

Abstract

INTRODUCTION
Highly purified RNA polymerase isolated from T4 phage-infected E. coli (T4 enzyme) contains a small amount (about 0.2 equivalent) of the sigma subunit and four new T4-specific polymerase binding proteins (Stevens 1972Stevens 1974; Horvitz 1973; Ratner 1974). When the enzyme is subjected to phosphocellulose column chromatography, the sigma (MW 95,000) fraction obtained also contains the small binding proteins of molecular weight 12,000 and 10,000, and the core enzyme fraction contains the binding proteins of molecular weight 15,000 and 22,000. We have recently described the properties of the sigma fraction (Stevens 1973Stevens 1974; Stevens and Rhoton 1975) as follows: The sigma fraction poorly stimulates core enzyme fractions except in the presence of Triton; with T4 DNA as a template, the sigma fraction inhibits the activity of core enzyme plus host sigma fractions, particularly in the presence of salt. In our work, further purification of the sigma subunit by gradient centrifugation showed that the fractions containing sigma also contain the polymerase binding protein of molecular weight 10,000 (10K). This protein could be separated from sigma by including 6 M urea in the gradient centrifugation solutions. The fractions containing the 10K protein had the salt-promoted inhibitory activity towards host sigma-stimulated core enzyme fractions. In this paper I report the separation of the sigma and 10K fractions by agarose gel chromatography in the presence of 6 M urea and describe the properties of the two separated fractions.

MATERIALS AND METHODS
T4 core enzyme and T4 sigma were prepared from E. coli cells infected...