Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.
Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.
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DOI：
10.1101/gad.285395.116
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发表时间：
2016-09-15
影响因子：
10.5
通讯作者：
Tjian R
中科院分区：
文献类型：
--
作者：
Zhang Z;English BP;Grimm JB;Kazane SA;Hu W;Tsai A;Inouye C;You C;Piehler J;Schultz PG;Lavis LD;Revyakin A;Tjian R
In this study, Zhang et al present a single-molecule imaging-based dynamic analysis of human RNA polymerase II preinitiation complex (PIC) assembly. They established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented by live-cell imaging and performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. 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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