Role of the mammalian transcription factors IIF, IIS, and IIX during elongation by RNA polymerase II.
We have used a recently developed system that allows the isolation of complexes competent for RNA polymerase II elongation (E. Bengal, A. Goldring, and Y. Aloni, J. Biol. Chem. 264:18926-18932, 1989). Pulse-labeled transcription complexes were formed at the adenovirus major late promoter with use of HeLa cell extracts. Elongation-competent complexes were purified from most of the proteins present in the extract, as well as from loosely bound elongation factors, by high-salt gel filtration chromatography. We found that under these conditions the nascent RNA was displaced from the DNA during elongation. These column-purified complexes were used to analyze the activities of different transcription factors during elongation by RNA polymerase II. We found that transcription factor IIS (TFIIS), TFIIF, and TFIIX affected the efficiency of elongation through the adenovirus major late promoter attenuation site and a synthetic attenuation site composed of eight T residues. These factors have distinct activities that depend on whether they are added before RNA polymerase has reached the attenuation site or at the time when the polymerase is pausing at the attenuation site. TFIIS was found to have antiattenuation activity, while TFIIF and TFIIX stimulated the rate of elongation. In comparison with TFIIF, TFIIS is loosely bound to the elongation complex. We also found that the activities of the factors are dependent on the nature of the attenuator. These results indicate that at least three factors play a major role during elongation by RNA polymerase II.
Bengal
E
E
Department of Molecular Genetics and Virology, Wiezmann Institute of Science, Rehovot, Israel.
Flores
O
O
Krauskopf
A
A
Reinberg
D
D
Aloni
Y
Y
D000260:Adenoviruses, Human / Q000235:genetics; D002474:Cell-Free System; D003001:Cloning, Molecular; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D012319:RNA Polymerase II / Q000378:metabolism*; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic; D014157:Transcription Factors / Q000502:physiology*; D035165:Transcription Factors, General; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors
D046911:Macromolecular Substances; D014157:Transcription Factors; D035165:Transcription Factors, General; D019473:Transcription Factors, TFII; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; D012319:RNA Polymerase II; C056604:transcription factor TFIIF