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

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.

CBP Regulates Recruitment and Release of Promoter-Proximal RNA Polymerase II

“…However, the C-terminal zinc-finger region in CBP is known to directly interact with TFIIB in vitro (Kwok et al, 1994), and our results support a model wherein CBP recruits TFIIB to promoters, possibly by this direct interaction. By recruiting TFIIB to promoters, CBP likely stimulates PIC assembly and Pol II recruitment, consistent with a highly dynamic TFIIB-promoter interaction (Zhang et al, 2016). Despite reduced Pol II levels in the promoter-proximal region after CBP inhibition, pausing is not completely eliminated and the pausing index increases, not decreases, indicating that CBP also modulates promoter escape.…”

p53 Dynamically Directs TFIID Assembly on Target Gene Promoters

“…Thus, dynamic interactions between p53, TFIID, and promoter DNA were characterized via a single-molecule colocalization assay using total internal reflection fluorescence (TIRF) microscopy ( Fig. 1A) (21,22). Over the first 15 min of binding, quantum dot (Qdot)-labeled TFIID (QD-TFIID) associated with only 1.5% of the Cy3/Cy5-labeled native wild-type hdm2 promoter DNA in the absence of p53 ( Fig.…”

“…To assess the impact of the core promoter in p53-mediated TFIID/DNA binding, mutations (i.e., the mutant core) known to weaken TFIID binding and inhibit transcription (22,34) were exploited. Upon addition of p53, TFIID's association with the mutant core template was further reduced compared to that seen with the wild-type promoter (Fig.…”

“…A recent study utilizing real-time single-molecule fluorescence microscopy provided the insight that TFIID's interaction with a synthetic core promoter DNA tightly correlates with productive transcription (21). Another single-molecule report uncovered TFIIB's dynamic binding to TFIID at the promoter (22). Additional live-cell studies also suggested that the PIC composition is extraordinarily dynamic, with the loading and promoter escape of RNA polymerase II occurring approximately every 4 to 9 s during bursts of transcription (23).…”

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

“…Simulations which embody diffusion and binding suggest that multivalent TFs could, in principle, facilitate intersegment transfer [9]. Previously, single-molecule fluorescence microscopy has been used to study TF localization in living cells across a range of model organisms, including bacteria, yeast and multi-cellular organisms [10–16]. Many studies suggest complexities in diffusion and binding [4,11,12,15,17] which may include intersegmental transfer [4,11,17].…”