Characterization of an inhibitor causing potassium chloride sensitivity of an RNA polymerase from T4 phage-infected Escherichia coli.

The nature of the inhibition by salt (KCl) of DNA-dependent RNA polymerase from T4 phage-infected Escherichia coli (T4 enzyme) was studied using holoenzyme preparations, core enzyme and sigma fractions obtained by phosphocellulose column chromatography, and sigma fractions further purified by gradient centrifugation in the presence and absence of 6 M urea. We showed with holoenzyme preparations that salt inhibits the formation of rifampicin-resistant preinitiation complexes. The inhibition was considerably reduced when a nonionic detergent (particularly of the Triton series) was included in the reaction mixtures. With T4 core enzyme and T4 sigma fractions together with the same fractions from uninfected cells (host enzyme fractions) and different DNA templates, we showed that the T4 sigma fraction plays a role in the salt-sensitive activity with T4 DNA. The salt sensitivity of the T4 sigma fraction was antagonized by Triton; it was not a function of sigma fractions isolated from phage cultures infected in the presence of chloramphenicol. As reported previously (Stevens, A. (1973), Biochem. Biophys. Res. Commun. 54, 488), the T4 sigma fraction inhibited the activity of host sigma when they were present together in reaction mixtures, particularly in the presence of salt. T4 sigma further purified by centrifugation in glycerol gradients had the same properties as the cruder fraction, and the T4-specific polypeptide of mol wt 10000 (Stevens, A. (1972), Proc. Natl. Acad. Sci. U.S.A. 69, 603) was found in the same fractions. If the glycerol gradients contained 6 M urea, the mol wt 10000 polypeptide was separated from the salt-stimulated sigma. Fractions containing the small polypeptide could be added back to produce the salt-inhibitory effects. The inhibitory activity of both the crude sigma fraction and the fractions containing the small polypeptide was inactivated at 65 degrees C. The results suggest that the mol wt 10000 protein is a salt-promoted inhibitor, but the small amounts of it which are present in purified fractions of the T4 enzyme have not yet allowed its isolation in large enough quantities to permit a detailed study of its properties.