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

Significance

After RNA synthesis is initiated by RNA polymerase II (pol II) and general transcription factors (GTFs), pol II dissociates from GTFs and promoter DNA and undergoes the transition to productive elongation. This transition is generally referred to as promoter escape and is a major rate-limiting step at many mRNA genes, but key details have remained unclear. We have developed an in vitro reconstituted transcription system that has high initiation efficiency, which allowed us to dissect the timing of this transition. We found that the formation of the initially transcribing complex, containing pol II, general transcription factors, and a nascent transcript, is long persisting and that 2 elongation factors conserved across eukaryotes have robust and positive biochemical activities on this transition.

Abstract

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.