Purification and characterization of ternary complexes containing accurately initiated RNA polymerase II and less than 20 nucleotides of RNA.
We have previously demonstrated that transcription of the adenovirus type 2 (Ad2) late promoter in vitro under UTP-limiting conditions results in pauses by the elongating RNA polymerase II between positions +6 and +17. We report here the purification of complexes between the paused RNA polymerase and a 260 base-pair Ad2 promoter-bearing DNA fragment. The procedure involves sedimentation through sucrose gradients, electrophoresis in agarose gels, and electroelution from the gels; the final complex pool is completely active in chain elongation. We observe a sharp discontinuity in complex stability during purification as a function of the number of bases added to the growing chains: complexes in which the polymerase has added more than ten bases are stable and are active in chain elongation even after the electroelution step, whereas complexes containing seven or fewer bases dissociate very easily. When purified complexes are extensively digested with proteinase K their electrophoretic mobility is increased considerably, yet they remain fully active in chain elongation. If purified complexes are digested with DNase I their electrophoretic mobility does not change. When the nuclease-treated complexes are allowed to continue chain elongation, they are able to add approximately 20 more bases to the nascent chains.
D000260:Adenoviruses, Human / Q000235:genetics; D002499:Centrifugation, Density Gradient; D004279:DNA, Viral / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005814:Genes, Viral; D009876:Operon; D012319:RNA Polymerase II / Q000235:genetics / Q000302:isolation & purification; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D014158:Transcription, Genetic
D004279:DNA, Viral; D012367:RNA, Viral; D012319:RNA Polymerase II