The capping enzyme facilitates promoter escape and assembly of a follow-on preinitiation complex for reinitiation.

After synthesis of a short nascent RNA, RNA polymerase II (pol II) dissociates general transcription factors (GTFs; TFIIA, TFIIB, TBP, TFIIE, TFIIF, and TFIIH) and escapes the promoter, but many of the mechanistic details of this process remain unclear. Here we developed an in vitro transcription system from the yeast Saccharomyces cerevisiae that allows conversion of the preinitiation complex (PIC) to bona fide initially transcribing complex (ITC), elongation complex (EC), and reinitiation complex (EC+ITC). By biochemically isolating postinitiation complexes stalled at different template positions, we have determined the timing of promoter escape and the composition of protein complexes associated with different lengths of RNA. Almost all of the postinitiation complexes retained the GTFs when pol II was stalled at position +27 relative to the transcription start site, whereas most complexes had completed promoter escape when stalled at +49. This indicates that GTFs remain associated with pol II much longer than previously expected. Nevertheless, the long-persisting transcription complex containing RNA and all of the GTFs is unstable and is susceptible to extensive backtracking of pol II. Addition of the capping enzyme and/or Spt4/5 significantly increased the frequency of promoter escape as well as assembly of a follow-on PIC at the promoter for reinitiation. These data indicate that elongation factors play an important role in promoter escape and that ejection of TFIIB from the RNA exit tunnel of pol II by the growing nascent RNA is not sufficient to complete promoter escape.

Def1 interacts with TFIIH and modulates RNA polymerase II transcription.

The DNA damage response is an essential process for the survival of living cells. In a subset of stress-responsive genes in humans, Elongin controls transcription in response to multiple stimuli, such as DNA damage, oxidative stress, and heat shock. Yeast Elongin (Ela1-Elc1), along with Def1, is known to facilitate ubiquitylation and degradation of RNA polymerase II (pol II) in response to multiple stimuli, yet transcription activity has not been examined. We have found that Def1 copurifies from yeast whole-cell extract with TFIIH, the largest general transcription factor required for transcription initiation and nucleotide excision repair. The addition of recombinant Def1 and Ela1-Elc1 enhanced transcription initiation in an in vitro reconstituted system including pol II, the general transcription factors, and TFIIS. Def1 also enhanced transcription restart from TFIIS-induced cleavage in a pol II transcribing complex. In the Δdef1 strain, heat shock genes were misregulated, indicating that Def1 is required for induction of some stress-responsive genes in yeast. Taken together, our results extend the understanding of the molecular mechanism of transcription regulation on cellular stress and reveal functional similarities to the mammalian system.