Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis
- Zhengjian Zhang1,
- Brian P. English1,
- Jonathan B. Grimm1,
- Stephanie A. Kazane2,
- Wenxin Hu1,
- Albert Tsai1,
- Carla Inouye3,4,
- Changjiang You5,
- Jacob Piehler5,
- Peter G. Schultz2,
- Luke D. Lavis1,
- Andrey Revyakin6 and
- Robert Tjian1,3,4
- 1Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA;
- 2Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037 USA;
- 3Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA;
- 4Li Ka Shing Center for Biomedical and Health Sciences, University of California at Berkeley, Berkeley, California 94720, USA;
- 5Department of Biology, University of Osnabrück, 49076 Osnabrück, Germany;
- 6Department of Molecular and Cell Biology, University of Leicester, Leicester LE1 9HN, United Kingdom
- Corresponding author: zhengjianzhang{at}gmail.com
Abstract
Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A “step-wise” preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB–promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II–TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.
Keywords
- single-molecule
- fluorescence imaging
- dynamic analysis
- transcription
- preinitiation complex
- in vitro reconstitution
Footnotes
-
Supplemental material is available for this article.
-
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.285395.116.
-
Freely available online through the Genes & Development Open Access option.
- Received June 11, 2016.
- Accepted September 1, 2016.
This article, published in Genes & Development, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.