A Protein Factor from Rat‐Liver Tissue Enhancing the Transcription of Native Templates by Homologous RNA Polymerase B

Abstract

A protein factor which stimulates the transcription of native templates catalyzed by enzyme B from rat liver has been isolated from the cytoplasm of this tissue. It has been purified extensively by chromatography through DEAE‐cellulose and CM‐cellulose, hydroxylapatite, gel‐filtration, rechromatography on DEAE‐cellulose and isoelectrofocussing. The protein is of low molecular weight (±30000) with an isoelectric point of 9.6. This factor predominantly stimulates enzyme B (±500%) without markedly effecting other RNA polymerase species from this tissue. Experiments have been adressed to the question concerning the mechanism through which this phenomenon operates. The stimulation manifests itself without noticeable time‐lag, immaterial of whether the factor is added before or after initiation of RNA synthesis and approximate estimation of the initiation complexes formed with or without factor, measured by direct filtration on millipore filters, lends no proof to the assumption that initiation is elevated. The degree of stimulation, measured on a template blocked by actinomycin D, thus allowing initiation but limiting propagation, is markedly diminished. This observation, taken together with the pronounced sensitivity of the factor‐induced RNA synthesis toward pyrophosphate, an inhibitor of phosphodiester bond formation, suggests that the observed effect operates through the chain‐elongation step. This assumption is supported by the fact that K s , determinations for CTP are not altered by the factor in the face of a greatly enhanced value for V , suggesting that the rate of chain propagation is raised. Experiments in which the 5′ termini of the RNA were labelled with purine nucleoside [γ‐ 32 P] triphosphates show that the average chain length of the product is increased through the action of the stimulatory protein. The factor‐induced stimulation is most pronounced on native templates of high molecular weight and it is possible that the protein studied aids in the unwinding process of such a DNA or enhances the rate of chain propagation in some other way.