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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.
Highlights
Transcription initiation by eukaryotic RNA polymerase II (Pol II) requires the coordinated action of Pol II and at least six general transcription factors (GTFs; i.e., TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) at promoters of proteinencoding genes (Thomas and Chiang 2006)
Since TFIID promoter binding is generally considered the first step in preinitiation complex (PIC) assembly, we began by visualizing their interaction with a TATA-box-containing promoter DNA template (Fig. 1A)
The ability of TFIID to discriminate between promoter DNA and the control fragment was much greater than what was observed for TATA-binding protein (TBP)
Summary
Transcription initiation by eukaryotic RNA polymerase II (Pol II) requires the coordinated action of Pol II and at least six general transcription factors (GTFs; i.e., TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) at promoters of proteinencoding genes (Thomas and Chiang 2006). Classical biochemical studies have established a “step-wise” model for the assembly of a Pol II transcription preinitiation complex (PIC) (Buratowski et al 1989) In this model, the core promoter recognition factor—TFIID, composed of the TATA-binding protein (TBP) and ∼14 TBP-associated factors (TAFs)—is the first factor to bind promoter DNA (Albright and Tjian 2000; Matangkasombut et al 2004). We found that TFIID- and TFIIA-dependent TFIIB binding is transient, with a residence time of ∼1.5 sec, which becomes stabilized only after a specific interaction with Pol II–TFIIF, indicating a transition to a functional PIC. We further confirmed these in vitro findings by live-cell single-molecule imaging. Our studies underscore the advantages of superresolution dynamic imaging studies to uncover previously undetected mechanisms regulating complex reactions such as those taking place during Pol II PIC assembly
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