Characterization of a protein factor stimulating RNA synthesis by DNA-dependent RNA polymerase II from plant cell cultures.

Abstract

During purification of DNA-dependent RNA polymerase II (or B) from cell cultures of parsley a protein fraction was separated by phosphocellulose chromatography which enhanced RNA synthesis in the presence of native homologous DNA. This 'stimulatory factor' was characterized in respect to some effects on the reaction catalyzed by RNA polymerase II. In the presence of the factor the metal ion requirements as well as the ionic strength for optimal RNA synthesis were markedly changed; addition of the factor to RNA polymerase II purified by cellulose chromatography restored those enzyme properties which had apparently changed upon this purification step. The chain length of the RNA product synthesized is favouring the view that the factor acts mainly by stabilizing the elongation step during transcription. The stimulatory factor was further purified by several steps of column chromatography. As derived from the results of gel electrophoresis under denaturing conditions the factor consists of several small polypeptides. Those of Mr = 26000, 25000 and 14000 apparently have counterparts among the smaller subunits of highly purified RNA polymerase II from parsley cells. Another polypeptide of the factor, with Mr = 30000, was only found in those preparations of RNA polymerase II which had not been subjected to phosphocellulose chromatography.