Elongation by Drosophila RNA polymerase II. Transcription of 3'-extended DNA templates.
RNA polymerase II will efficiently initiate transcription on linear duplex DNA which has been extended at its 3' ends by the addition of short stretches of polydeoxycytidine (Kadesch, T. R., and Chamberlin, M. J. (1982) J. Biol. Chem. 257, 5286-5295). We have used such dC-tailed templates to identify factors affecting elongation by Drosophila RNA polymerase II (Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1987) J. Biol. Chem. 262, 3244-3255). While studying these factors we have observed two unexpected characteristics of transcription of the tailed templates. First, we found that RNA polymerase II encountered a strong pause site after the incorporation of 14 nucleotides. This pausing was observed on all templates examined and with RNA polymerase II from a variety of sources. In addition, we found that ammonium ions markedly stimulated the polymerase, increasing both the efficiency with which the enzyme left the 14 base pause site and the subsequent rate of elongation. A factor previously shown to affect transcription of dC-tailed templates (factor 4, Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1987) J. Biol. Chem. 262, 3244-3255) was found to cause transcript displacement and to stimulate the elongation rate approximately 2-fold. This factor copurified with an RNase H activity, and a model is presented for the mechanism of transcript displacement by RNase H. The observations presented here form a basis for further analysis of RNA polymerase II elongation and its modulation by transcription factors. They should also aid in the interpretation of other experiments in which dC-tailed templates are used.
Sluder
A E
AE
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Price
D H
DH
Greenleaf
A L
AL
D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D004247:DNA / Q000378:metabolism*; D003841:Deoxycytidine / Q000378:metabolism; D004330:Drosophila / Q000201:enzymology*; D004722:Endoribonucleases / Q000378:metabolism; D007700:Kinetics; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism*; D016914:Ribonuclease H; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000187:drug effects
D009692:Nucleic Acid Heteroduplexes; D009711:Nucleotides; D003841:Deoxycytidine; D012313:RNA; D011189:Potassium Chloride; D004247:DNA; D012319:RNA Polymerase II; D004722:Endoribonucleases; D016914:Ribonuclease H; D012259:Ribonuclease, Pancreatic; D000645:Ammonium Sulfate