Studies on transcription of 3'-extended DNA templates by mammalian RNA polymerase II. Partial purification and characterization of a factor from HeLa cells that facilitates renaturation of the DNA template

Publisher Website
Google Scholar
Abstract
Transcription by purified mammalian RNA polymerase II in vitro leads to extensive formation of DNA-RNA hybrids between nascent RNA and the template DNA strand. This is especially clear during transcription of 3''-extended (dC-tailed) DNA templates where the nontranscribed DNA strand is progressively displaced as transcription proceeds. Addition of small amounts of a HeLa cell extract to such a transcription system enhances renaturation of the template DNA and displacement of the nascent RNA, as measured by the sensitivity of the RNA to pancreatic RNase. Using this latter assay, a protein factor (renaturase) was purified 250-fold from HeLa cell extracts using chromatography on DEAE-cellulose, DNA-cellulose and hydroxylapatite. Renaturase preparations facilitate complete renaturation of the template DNA duplex during transcription by RNA polymerase II and lead to concurrent displacement of the nascent RNA. Current preparations are free from all but traces of DNase or RNase. The active component has a MW of about 30,000 as estimated by preparative density gradient sedimentation. The structure of transcribing RNA polymerase II complexes was examined in the presence and absence of renaturase, using the EM and the Williams polylysine technique. In the presence of renaturase, the DNA template is fully renatured, and a ternary complex in which the nascent RNA is displaced during transcription is seen. A number of nucleic acid binding proteins were tested for renaturase activity and are inactive, including Echerichia coli single-stranded binding protein, .rho. factor, recA protein, HU protein, T4 phage gene 32 protein, histone H2A and high mobility group proteins, 15S and 30S heterogeneous nuclear ribonucleoprotein particles, avian myeloblastosis virus reverse transcriptase and Xenopus topoisomerase I.