Abstract
Protein purification and depletion studies were used to determine the major stable forms of RNA polymerase II (Pol II) complexes found in Saccharomyces cerevisiae nuclear extracts. About 50% of Pol II is found associated with the general transcription factor TFIIF (Pol II-TFIIF), and about 20% of Pol II is associated with Mediator (Pol-Med). No Pol II-Med-TFIIF complex was observed. The activity of Pol II and the purified Pol II complexes in transcription initiation and reinitiation was investigated by supplementing extracts depleted of either total Pol II or total TFIIF with purified Pol II or the Pol II complexes. We found that all three forms of Pol II can complement Pol II-depleted extracts for transcription initiation, but Pol II-TFIIF has the highest specific activity. Similarly, Pol II-TFIIF has a much higher specific activity than TFIIF for complementation of TFIIF transcription activity. Although the Pol II-TFIIF and Pol II-Med complexes were stable when purified, we found these complexes were dynamic in extracts under transcription conditions, with a single polymerase capable of exchanging bound Mediator and TFIIF. Using a purified system to examine transcription reinitiation, we found that Pol II-TFIIF was active in promoting multiple rounds of transcription while Pol II-Med was nearly inactive. These results suggest that both the Pol II-Med and Pol II-TFIIF complexes can be recruited for transcription initiation but that only the Pol II-TFIIF complex is competent for transcription reinitiation.