Real-Time Observation of the Initiation of RNA Polymerase II Transcription

Biochemical and structural studies have shown that the initiation of RNA polymerase II (pol II) transcription proceeds in the following stages: assembly of pol II with general transcription factors (GTFs) and promoter DNA in a “closed” preinitiation complex (PIC)(1,2); unwinding about 15 bp of the promoter DNA to form an “open” complex(3,4); scanning downstream to a transcription start site; synthesis of a short transcript, believed to be about 10 nucleotides; and promoter escape. We have assembled a 32-protein, 1.5 megadalton PIC(5) derived from Saccharomyces cerevisiae and observed subsequent initiation processes in real time with optical tweezers(6). Contrary to expectation, scanning driven by transcription factor IIH (TFIIH)(7-12) entailed the rapid opening of an extended bubble, averaging 85 bp, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.