Transcription complexes synthesizing attenuated RNA can serve as a model system for analyzing elongation factors.
In vitro transcription elongation, in HeLa whole cell extract, is partially blocked at the Ad2 and synthetic (8 Ts) attenuation sites when transcription elongation is performed at increasing KCl concentrations (0.2-0.5 M KCl) but not at the standard KCl concentration (50 mM). Furthermore, if briefly initiated transcription complexes are chromatographed on Sephacryl column at increased KCl concentration and the complexes, which are eluted in the void volume, are diluted to 50 mM KCl, they will elongate a substantial fraction of the briefly initiated nascent RNA into attenuated RNA. The addition of a crude preparation of a stimulatory factor to the complexes diluted to 50 mM KCl prevents the elongation blocks. Based on the notion that putative elongation factors dissociate from the transcription complexes at increasing KCl concentrations and in the Sephacryl column they elute after the initiation complexes, we suggest that the column-purified transcription complexes, which have been depleted of elongation factors, can serve as a model system for determining the activities of factors during elongation.
Bengal
E
E
Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot, Israel.
Goldring
A
A
Aloni
Y
Y
D000260:Adenoviruses, Human / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000187:drug effects
D004279:DNA, Viral; D012367:RNA, Viral; D011189:Potassium Chloride