c-Jun Binds the N Terminus of Human TAFII250 to Derepress RNA Polymerase II Transcription in Vitro

p53 is a central regulator that turns on vast gene networks to maintain cellular integrity in the presence of various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key issue, we have undertaken an integrated approach involving single-molecule fluorescence microscopy, single-particle cryo-electron microscopy, and biochemistry. Our real-time single-molecule imaging data demonstrate that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIID's ability to bind DNA by stabilizing TFIID contacts with both the core promoter and a region within p53's response element. Analysis of single-molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIID's conversion to a rearranged DNA binding conformation is enhanced in the presence of DNA and p53. Notably, TFIID's interaction with DNA induces p53 to rapidly dissociate, which likely leads to additional rounds of p53-mediated recruitment of other basal factors. Collectively, these findings indicate that p53 dynamically escorts and loads TFIID onto its target promoters. KEYWORDS p53, TFIID, transcription, structure, single-molecule microscopy M ore than 50% of cancer patients harbor p53 mutations, highlighting the essential role of this protein in tumor suppression (1). To properly maintain genomic stability, p53 induces vast gene networks involved in diverse cellular pathways, including the cell cycle arrest, apoptosis, and DNA repair pathways (2). In response to various stress stimuli, p53 acts as a transcriptional activator that specifically binds consensus response elements (REs; two 10-base-pair half-sites [RRRCWWGYYY]) within its target genes to directly stimulate gene expression (2). p53 utilizes its ability to nonspecifically bind and slide along the DNA to expedite the search for target REs (3). Upon recognition of its response genes, p53 facilitates transcription at least in part by targeting the TFIID-mediated transcription machinery to the promoter (4-7). TFIID is composed of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). TFIID recognizes and binds multiple core promoter elements (e.g., the TATA box, the initiator, and downstream core promoter elements [DPE]) surrounding the transcription start site (TSS) (8). Once bound, TFIID serves as a central scaffold for six basal factors, including RNA polymerase II, to form a preinitiation complex (PIC) directing transcription initiation. Importantly, without the assistance of activators such as p53, TFIID's core promoter recognition is weak and rate limiting for transcription initiation due to inefficient PIC assembly (9-16). As TFIID is responsible for transcription of at least ϳ90% of proteincoding genes (17), unraveling how p...

Section: Discussionmentioning

confidence: 99%

p53 Dynamically Directs TFIID Assembly on Target Gene Promoters

Coleman

Qiao

Singh

et al. 2017

“…Reversibility of this concave inhibition would be a key feature to allow TBP to bind DNA. It has been proposed that transcriptional activators like c-Jun, VP16, GAL4, and EBNA2 may regulate dTAF II 230/hTAF II 250 inhibition of TBP binding to DNA (30,32,35,41). Might hTAF II 170 inhibition of TBP be similarly modulated?…”

Section: Discussionmentioning

confidence: 99%

TAF_II170 Interacts with the Concave Surface of TATA-Binding Protein To Inhibit Its DNA Binding Activity

Pereira

Knaap

Boom

et al. 2001

The human RNA polymerase II transcription factor B-TFIID consists of TATA-binding protein (TBP) and the TBP-associated factor (TAF) TAF II 170 and can rapidly redistribute over promoter DNA. Here we report the identification of human TBP-binding regions in human TAF II 170. We have defined the TBP interaction domain of TAF II 170 within three amino-terminal regions: residues 2 to 137, 290 to 381, and 380 to 460. Each region contains a pair of Huntington-elongation-A subunit-Tor repeats and exhibits species-specific interactions with TBP family members. Remarkably, the altered-specificity TBP mutant (TBP AS ) containing a triple mutation in the concave surface is defective for binding the TAF II 170 amino-terminal region of residues 1 to 504. Furthermore, within this region the TAF II 170 residues 290 to 381 can inhibit the interaction between Drosophila TAF II 230 (residues 2 to 81) and TBP through competition for the concave surface of TBP. Biochemical analyses of TBP binding to the TATA box indicated that TAF II 170 region 290-381 inhibits TBP-DNA complex formation. Importantly, the TBP AS mutant is less sensitive to TAF II 170 inhibition. Collectively, our results support a mechanism in which TAF II 170 induces high-mobility DNA binding by TBP through reversible interactions with its concave DNA binding surface.Initiation of mRNA synthesis by RNA polymerase (pol) II requires the basal transcription factors TFIID, TFIIB, TFIIE, TFIIF, and TFIIH. Recognition of the TATA box within core pol II promoters by TFIID initiates the assembly of a functional pol II preinitiation complex. TFIID is a multisubunit complex consisting of TATA-binding protein (TBP) and at least 10 TBP-associated factors (TAF II s) ranging in size from 15 to 250 kDa. TAF II s play several roles in transcriptional regulation, serving as coactivators, promoter selectivity factors, or enzymes that modify surrounding transcriptional proteins (3). The TBP subunit recognizes the TATA box forming the TBP-TATA complex (24). Structural studies of the TBP-TATA complex indicate that the ␤ sheets of TBP form a concave undersurface which contacts the minor groove of the TATA box (28,29).Of relevance to this study is that the largest TAF II subunit of TFIID (dTAF II 230 in Drosophila, hTAF II 250 in humans, and yTAF II 145/130 in yeast), can inhibit TBP binding to the TATA box (11,31,33). dTAF II 230 achieves this by interacting with the concave DNA binding surface of TBP (see references cited above). Residues 2 to 81 (called TAND I) undergo a disorderto-order transition upon interaction with TBP. The induced structure of TAND I mimics the structure of the TATA box when bound to TBP (34). A second N-terminal inhibitory domain (TAND II; residues 82 to 156) blocks the TBP-TFIIA interaction by competitively binding to helix 2 (H2) on the convex surface of TBP (10). The orthologous TAF II subunits from yeast and humans contain regions which are functionally equivalent to TAND I and TAND II despite a low degree of primary sequence similarity (33).TAF II -contain...

“…An increasing amount of evidence suggests that the TAND participates in transcriptional activation even though it is not absolutely required (5,6,8,14,15,29). For example, the TBP-inhibitory activity of human TAF1 can be reduced in vitro by the action of the AD (14,15).…”

mentioning

confidence: 99%

Autonomous Function of the Amino-Terminal Inhibitory Domain of TAF1 in Transcriptional Regulation

Takahata

Koyamada

Kawaichi

et al. 2004

The general transcription factor TFIID is composed of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). TFIID mediates the transcriptional activation of a subset of eukaryotic promoters. The N-terminal domain (TAND) of TAF1 protein (Taf1p) inhibits TBP by binding to its concave and convex surfaces. This study examines the role of the TAND in transcriptional regulation and tests whether the TAND is an autonomous regulator of TBP. The TAND binds to and regulates TBP function when it is fused to the amino or carboxy terminus of Taf1p, the amino or carboxy terminus of Taf5p, or the amino terminus of Taf11p. However, a carboxy-terminal fusion of the TAND and Taf11p is not compatible with several other TAF proteins, including Taf1p, in the TFIID complex. These results indicate that there is no or minimal geometric constraint on the ability of the TAND to function normally in transcriptional regulation as long as TFIID assembly is secured.Transcription of eukaryotic protein-coding genes is regulated by the concerted action of a number of distinct transcription factors (reviewed in references 34 and 38). These transcription factors are recruited to the promoter region in a precise gene-specific order (reviewed in reference 10). Transcription factor IID (TFIID) is a general transcription factor complex that is composed of the TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs) (39, 41). It recognizes and binds to several core promoter elements and is thought to be a platform for the subsequent assembly of the preinitiation complex (reviewed in reference 3). TFIID also plays a role in transcriptional activation (reviewed in references 3 and 34). Extensive studies using in vitro reconstitution systems have shown that the involvement of the TAFs that make up TFIID in transcriptional activation can vary depending on the experimental conditions, e.g., whether mediator components are included and/or nucleosomal DNA is used as a template (16,35,43). In good agreement with these observations, genetic studies of yeast revealed that the requirement for TAFs in in vivo transcription depends on the promoter structures of the target genes (reviewed in reference 13).Yeast promoters are classified as TAF dependent (TAF dep ) or TAF independent (TAF ind ) (23, 25). In vivo cross-linking and chromatin immunoprecipitation analyses indicate that TAFs interact infrequently with TAF ind promoters but frequently with TAF dep promoters at an approximately equimolar ratio to TBP. In the case of the TAF dep promoters of ribosomal protein (RP) genes, TAF recruitment is directed by upstream activating sequences (UAS) but not by the core promoter and it does not depend on the function of TBP and pol II (26, 32).In contrast, the TAF ind UAS in the PGK1 and PYK1 promoters do not efficiently recruit TAFs to TAF dep or TAF ind core promoters (32). These observations indicate that an activator(s) bound to a TAF dep UAS may specifically interact with TAF(s) and recruit TFIID onto the core promoter. In addition, there are some f...