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

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.

Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription

“…The kinetics of RNApII basal transcription initiation (i.e., initiation that occurs in the absence of activators) has been previously examined in vitro, including by single-molecule methods (41,42,(44)(45)(46). Such studies typically observe transcription from only a small fraction of template molecules, although this can be greatly improved by pre-formation of PICs before initiating RNA synthesis (47)(48)(49). Here we focused on the dynamics of activated transcription, which is thought to be the dominant mode of gene expression in vivo.…”

Interplay of mRNA capping and transcription machineries

“…Ceg1 stimulates early elongation [74,77], with its mutants being defective in this process. The latest findings show that the Cet1-Ceg1 complex and DSIF stimulate Pol II promoter escape and transition to elongation [78].…”

Insight into promoter clearance by RNA polymerase II

“…Fuijwara et al (5) assembled complexes with wellcharacterized, very highly purified yeast GTFs and yeast pol II and then advanced the complexes into transcription in reactions with labeled NTPs but lacking GTP. Most of their studies utilized templates in which the first G on the nontemplate strand is located at either 27 bp (+27 template) or 49 bp (+49 template) downstream of the transcription start region.…”

“…Reactions were resolved on glycerol gradients and individual fractions were assayed for labeled RNA and protein content. As an important additional feature, Fuijwara et al (5) explored the effects of adding the yeast capping enzymes or elongation factors to the reactions prior to separation on gradients.…”

“…The mechanism through which the PIC/initiating complex transitions to the committed elongation complex represents a major unanswered challenge in transcription biochemistry. In PNAS, Fujiwara et al (5) report the application of a significantly improved yeast pol II in vitro transcription system to provide important insight into pol II promoter clearance.…”