Journal Article

Real-time observation of the initiation of RNA polymerase II transcription

Meng, Cong A., Murakami, Kenji, Kornberg, Roger D., Fazal, Furqan M., Block, Steven M.

2015

Overview

A single-molecule optical tweezer assay is developed to monitor transcription initiation in eukaryotic RNA polymerase II in real-time, making use of a highly purified preinitiation complex (PIC) from yeast; observations show that a large bubble is opened up in the DNA template during initiation, driven by the TFIIH helicase that forms part of the PIC, along with synthesis of an extended transcript before the transition from transcription initiation into elongation. Transcription initiation observed in real time This paper reports on the development of a single-molecule optical tweezer assay to monitor transcription initiation by eukaryotic RNA polymerase II in real-time, making use of a highly purified preinitiation complex (PIC) from yeast. Steven Block and colleagues find that a large bubble is opened up in the DNA template during initiation, driven by the TFIIH helicase that forms part of the PIC. This is accompanied by synthesis of an extended transcript prior to the transition from transcription initiation into elongation. These findings may also be relevant for higher eukaryotes, including metazoans. Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a ‘closed’ preinitiation complex (PIC) 1 , 2 ; unwinding of about 15 base pairs of the promoter DNA to form an ‘open’ complex 3 , 4 ; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC 5 derived from Saccharomyces cerevisiae , and observe subsequent initiation processes in real time with optical tweezers 6 . Contrary to expectation, scanning driven by the transcription factor IIH 7 , 8 , 9 , 10 , 11 , 12 involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/337/572 / 631/57/2265 / 82/83 / Base Pairing - genetics / Deoxyribonucleic acid / DNA / DNA - genetics / DNA - metabolism / Genetic research / Genetic transcription / Humanities and Social Sciences / letter / multidisciplinary / Optical Tweezers / Physiological aspects / Promoter Regions, Genetic - genetics / Proteins / RNA polymerase / RNA Polymerase II - chemistry / RNA Polymerase II - metabolism / RNA polymerases / RNA, Messenger - biosynthesis / RNA, Messenger - genetics / Saccharomyces cerevisiae - enzymology / Saccharomyces cerevisiae - genetics / Science / Studies / Transcription Factor TFIIH - metabolism / Transcription factors / Transcription Initiation, Genetic