Specific transcription of preformed nucleoprotein complexes, containing the adenovirus major late promoter, with a chromatographic fraction containing RNA polymerase II.
Incubation in a HeLa whole-cell extract converted a plasmid DNA containing the adenovirus type 2 major late promoter into ordered nucleoprotein complexes similar to those described for simian virus DNA [Sinha, S. N., Hellwig, R. J., Allison, D. P. & Niyogi, S. K. (1982) Nucleic Acids Res. 10, 5533-5552]. Purified nucleoprotein complexes containing the plasmid DNA were able to serve as template for accurate transcription in vitro. Use of such nucleoprotein complexes eliminated the need for addition of nontemplate DNA (poly[d(I-C)]) to transcription reaction mixtures with template concentrations too low to yield a detectable specific-transcription signal from "naked" DNA. Of the four fractions resulting from phosphocellulose column chromatography of the whole cell extract, only the fraction that contained the RNA polymerase II activity was needed to accurately transcribe these nucleoprotein complexes in the presence of human placental ribonuclease inhibitor. In contrast, specific transcription of naked template DNA under these conditions required at least one additional fraction containing specificity factors. These results show that the nucleoprotein complexes contain factor(s) needed for specific initiation of transcription and that these complexes may be useful in the purification and analysis of these factors.
D000260:Adenoviruses, Human / Q000235:genetics*; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D009698:Nucleoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011089:Polydeoxyribonucleotides / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000187:drug effects
D009698:Nucleoproteins; D011089:Polydeoxyribonucleotides; C031156:poly d(I-C); D012319:RNA Polymerase II