The gene encoding the large subunit of human RNA polymerase II

Abstract

As a first step to approach the structural and functional analysis of DNA-dependent RNA polymerase II (EC 2.7.7.8), we have isolated genomic sequences for the large subunit of the human enzyme. The sequences homologous to Drosophila RNA polymerase II large subunit sequences are present in the genome as single copy genes, when assayed at high stringency. The polypeptide information is encoded in a mRNA of 7.35 kilobases, as determined by Northern blot analysis. In vitro translation reveals a polypeptide of 220 kDa, similar in electrophoretic mobility to the largest subunit of the enzyme. A fusion-polypeptide synthesized in bacteria contains a region that cross-reacts with anti-RNA polymerase II antiserum. Antiserum directed against the purified fusion protein reacts with the large subunit of RNA polymerase II, whether in the intact IIA (220 kDa) or in the degraded IIB (180 kDa) forms. Moreover, the antifusion protein antibody inhibits not only the purified calf thymus RNA polymerase II activity but also specific RNA polymerase II transcription in a HeLa cell extract. Thus, the DNA fragment isolated contains structural and functional domains of the human RNA polymerase II large subunit.