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Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis

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 measurem...

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Autores principales: Zhang, Zhengjian, English, Brian P., Grimm, Jonathan B., Kazane, Stephanie A., Hu, Wenxin, Tsai, Albert, Inouye, Carla, You, Changjiang, Piehler, Jacob, Schultz, Peter G., Lavis, Luke D., Revyakin, Andrey, Tjian, Robert
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066616/
https://www.ncbi.nlm.nih.gov/pubmed/27798851
http://dx.doi.org/10.1101/gad.285395.116
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author Zhang, Zhengjian
English, Brian P.
Grimm, Jonathan B.
Kazane, Stephanie A.
Hu, Wenxin
Tsai, Albert
Inouye, Carla
You, Changjiang
Piehler, Jacob
Schultz, Peter G.
Lavis, Luke D.
Revyakin, Andrey
Tjian, Robert
author_facet Zhang, Zhengjian
English, Brian P.
Grimm, Jonathan B.
Kazane, Stephanie A.
Hu, Wenxin
Tsai, Albert
Inouye, Carla
You, Changjiang
Piehler, Jacob
Schultz, Peter G.
Lavis, Luke D.
Revyakin, Andrey
Tjian, Robert
author_sort Zhang, Zhengjian
collection PubMed
description 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.
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spelling pubmed-50666162016-10-31 Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis Zhang, Zhengjian English, Brian P. Grimm, Jonathan B. Kazane, Stephanie A. Hu, Wenxin Tsai, Albert Inouye, Carla You, Changjiang Piehler, Jacob Schultz, Peter G. Lavis, Luke D. Revyakin, Andrey Tjian, Robert Genes Dev Research Paper 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. Cold Spring Harbor Laboratory Press 2016-09-15 /pmc/articles/PMC5066616/ /pubmed/27798851 http://dx.doi.org/10.1101/gad.285395.116 Text en © 2016 Zhang et al.; Published by Cold Spring Harbor Laboratory Press http://creativecommons.org/licenses/by/4.0/ 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/.
spellingShingle Research Paper
Zhang, Zhengjian
English, Brian P.
Grimm, Jonathan B.
Kazane, Stephanie A.
Hu, Wenxin
Tsai, Albert
Inouye, Carla
You, Changjiang
Piehler, Jacob
Schultz, Peter G.
Lavis, Luke D.
Revyakin, Andrey
Tjian, Robert
Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis
title Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis
title_full Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis
title_fullStr Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis
title_full_unstemmed Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis
title_short Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis
title_sort rapid dynamics of general transcription factor tfiib binding during preinitiation complex assembly revealed by single-molecule analysis
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066616/
https://www.ncbi.nlm.nih.gov/pubmed/27798851
http://dx.doi.org/10.1101/gad.285395.116
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