Tfiih Generates a Six-Base-Pair Open Complex during Eukaryotic Transcription Initiation

Abstract

The initiation of messenger RNA transcription in Eukaryotes is directed by the formation of a megaDalton-sized multi-protein complex known as the pre-initiation complex (PIC). After PIC formation, double-stranded DNA is unwound to form a single-stranded DNA bubble and the template strand is loaded into the polymerase active site. Initial DNA opening is ATP-dependent and is catalyzed by Ssl2/XPB, a dsDNA translocase subunit of the basal transcription factor TFIIH. In yeast, transcription initiation proceeds through a scanning phase where downstream DNA is searched for optimal start-sites. Here, to test different models for initial DNA opening and start-site scanning, we measure the size of the DNA bubble formed by Saccharomyces cerevisiae PICs in real time using a single-molecule magnetic tweezers assay. We show that ATP hydrolysis by Ssl2 leads to the opening of a 6 base-pair (bp) DNA bubble that is expanded to 13 bp in the presence of NTPs. These observations support a two-step DNA opening model wherein ATP-dependent Ssl2 translocation leads to a 6 bp open complex which RNA polymerase II expands via NTP-dependent RNA transcription.