Purification of a cytosolic factor from rat liver that stimulates transcription in isolated nuclei and its action on purified RNA polymerase II-DNA system.
A cytosolic factor that stimulates transcription in isolated nuclei was purified approximately 4000-fold to near homogeneity from rat liver. The molecular weight of the factor was determined as 47 000 by SDS-polyacrylamide gel electrophoresis. The factor had no detectable deoxyribonuclease and protease activity but showed ribonuclease inhibitor activity. The factor could stimulate transcription in isolated nuclei by 50% at about 3.0 ng and the maximal stimulation was about 100%. When [gamma-S]ATP and [gamma-S]GTP were included in the reaction, the factor stimulated the synthesis of RNA which was able to bind to a mercury-Sepharose column and about 80% of the bound RNA was sensitive to a low concentration of alpha-amanitin. When heparin was added before initiation to preincubation mixture containing RNA polymerases II and DNA, a small but definite incorporation of [14C]UTP was observed. The factor alone had no stimulatory effect on the heparin-resistant incorporation of [14C]UTP but, in the presence of two rat liver nuclear fractions, phosphocellulose 0.5 and 1 M KCl step fractions, could stimulate the incorporation above the level with the combination of the two nuclear fractions. Antibody raised against the factor inhibited accurate transcription from the adenovirus 2 major late promoter in a nuclear lysate from Ehrlich ascites tumor cells, and the inhibition was neutralized by the factor.
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000502:physiology; D004247:DNA / Q000235:genetics*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008970:Molecular Weight; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D012319:RNA Polymerase II / Q000378:metabolism*; D051381:Rats; D014158:Transcription, Genetic
D011506:Proteins; D004247:DNA; D012319:RNA Polymerase II