Activity Of TATA-binding Protein Research Articles

SAGA and TFIID: Friends of TBP drifting apart

Transcription initiation constitutes a major checkpoint in gene regulation across all living organisms. Control of chromatin function is tightly linked to this checkpoint, which is best illustrated by the SAGA coactivator. This evolutionary conserved complex of 18–20 subunits was first discovered as a Gcn5p-containing histone acetyltransferase, but it also integrates a histone H2B deubiquitinase. The SAGA subunits are organized in a modular fashion around its central core. Strikingly, this central module of SAGA shares a number of proteins with the central core of the basal transcription factor TFIID. In this review I will compare the SAGA and TFIID complexes with respect to their shared subunits, structural organization, enzymatic activities and chromatin binding. I will place a special emphasis on the ancestry of SAGA and TFIID subunits, which suggests that these complexes evolved to control the activity of TBP (TATA-binding protein) in directing the assembly of transcription initiation complexes.

Core promoter-specific gene regulation: TATA box selectivity and Initiator-dependent bi-directionality of serum response factor-activated transcription

Gene-specific activation by enhancers involves their communication with the basal RNA polymerase II transcription machinery at the core promoter. Core promoters are diverse and may contain a variety of sequence elements such as the TATA box, the Initiator (INR), and the downstream promoter element (DPE) recognized, respectively, by the TATA-binding protein (TBP) and TBP-associated factors of the TFIID complex. Core promoter elements contribute to the gene selectivity of enhancers, and INR/DPE-specific enhancers and activators have been identified. Here, we identify a TATA box-selective activating sequence upstream of the human β-actin (ACTB) gene that mediates serum response factor (SRF)-induced transcription from TATA-dependent but not INR-dependent promoters and requires the TATA-binding/bending activity of TBP, which is otherwise dispensable for transcription from a TATA-less promoter. The SRF-dependent ACTB sequence is stereospecific on TATA promoters but activates in an orientation-independent manner a composite TATA/INR-containing promoter. More generally, we show that SRF-regulated genes of the actin/cytoskeleton/contractile family tend to have a TATA box. These results suggest distinct TATA-dependent and INR-dependent mechanisms of TFIID-mediated transcription in mammalian cells that are compatible with only certain stereospecific combinations of activators, and that a TBP-TATA binding mechanism is important for SRF activation of the actin/cytoskeleton-related gene family.

Cooperative action of NC2 and Mot1p to regulate TATA-binding protein function across the genome

Promoter recognition by TATA-binding protein (TBP) is an essential step in the initiation of RNA polymerase II (pol II) mediated transcription. Genetic and biochemical studies in yeast have shown that Mot1p and NC2 play important roles in inhibiting TBP activity. To understand how TBP activity is regulated in a genome-wide manner, we profiled the binding of TBP, NC2, Mot1p, TFIID, SAGA, and pol II across the yeast genome using chromatin immunoprecipitation (ChIP)-chip for cells in exponential growth and during reprogramming of transcription. We find that TBP, NC2, and Mot1p colocalize at transcriptionally active pol II core promoters. Relative binding of NC2alpha and Mot1p is higher at TATA promoters, whereas NC2beta has a preference for TATA-less promoters. In line with the ChIP-chip data, we isolated a stable TBP-NC2-Mot1p-DNA complex from chromatin extracts. ATP hydrolysis releases NC2 and DNA from the Mot1p-TBP complex. In vivo experiments indicate that promoter dissociation of TBP and NC2 is highly dynamic, which is dependent on Mot1p function. Based on these results, we propose that NC2 and Mot1p cooperate to dynamically restrict TBP activity on transcribed promoters.

Functional Silencing of TATA-binding Protein (TBP) by a Covalent Linkage of the N-terminal Domain of TBP-associated Factor 1

General transcription factor TFIID is comprised of TATA-binding protein (TBP) and TBP-associated factors (TAFs), together playing critical roles in regulation of transcription initiation. The TAF N-terminal domain (TAND) of yeast TAF1 containing two subdomains, TAND1 (residues 10-37) and TAND2 (residues 46-71), is sufficient to interact with TBP and suppress the TATA binding activity of TBP. However, the detailed structural analysis of the complex between yeast TBP and TAND12 (residues 6-71) was hindered by its poor solubility and stability in solution. Here we report a molecular engineering approach where the N terminus of TBP is fused to the C terminus of TAND12 via linkers of various lengths containing (GGGS)(n) sequence, (n = 1, 2, 3). The length of the linker within the TAND12-TBP fusion has a significant effect on solubility and stability (SAS). The construct with (GGGS)(3) linker produces the best quality single-quantum-coherence (HSQC) NMR spectrum with markedly improved SAS. In parallel to these observations, the TAND12-TBP fusion exhibits marked reduction of TBP function in binding to TAF1 as well as temperature sensitivity in in vivo yeast cell growth. Remarkably, the temperature sensitivity was proportional to the length of the linker in the fusions: the construct with (GGGS)(3) linker did not grow at 20 degrees C, while those with (GGGS)(1) and (GGGS)(2) linkers did. These results together indicate that the native interaction between TBP and TAND12 is well maintained in the TAND12-(GGGS)(3)-TBP fusion and that this fusion approach provides an excellent model system to investigate the structural detail of the TBP-TAF1 interaction.

Stimulation of Myc transactivation by the TATA binding protein in promoter-reporter assays

BackgroundThe c-Myc oncogenic transcription factor heterodimerizes with Max, binds specific DNA sites and regulates transcription. The role of Myc in transcriptional activation involves its binding to TRRAP and histone acetylases; however, Myc's ability to activate transcription in transient transfection assays is remarkably weak (2 to 5 fold) when compared to other transcription factors. Since a deletion Myc mutant D106-143 and a substitution mutant W135E that weakly binds TRRAP are still fully active in transient transfection reporter assays and the TATA binding protein (TBP) has been reported to directly bind Myc, we sought to determine the effect of TBP on Myc transactivation.ResultsWe report here a potent stimulation of Myc transactivation by TBP, allowing up to 35-fold transactivation of reporter constructs. Although promoters with an initiator (InR) element briskly responded to Myc transactivation, the presence of an InR significantly diminished the response to increasing amounts of TBP. We surmise from these findings that promoters containing both TATA and InR elements may control Myc responsive genes that require brisk increased expression within a narrow window of Myc levels, independent of TBP. In contrast, promoters driven by the TATA element only, may also respond to modulation of TBP activity or levels.ConclusionOur observations not only demonstrate that TBP is limiting for Myc transactivation in transient transfection experiments, but they also suggest that the inclusion of TBP in Myc transactivation assays may further improve the characterization of c-Myc target genes.

Structural and Functional Characterization on the Interaction of Yeast TFIID Subunit TAF1 with TATA-binding Protein

General transcription factor TFIID, consisting of TATA-binding protein (TBP) and TBP-associated factors (TAFs), plays a central role in both positive and negative regulation of transcription. The TAF N-terminal domain (TAND) of TAF1 has been shown to interact with TBP and to modulate the interaction of TBP with the TATA box, which is required for transcriptional initiation and activation of TATA-promoter operated genes. We have previously demonstrated that the Drosophila TAND region of TAF1 (residues 11–77) undergoes an induced folding from a largely unstructured state to a globular structure that occupies the DNA-binding surface of TBP thereby inhibiting the DNA-binding activity of TBP. In Saccharomyces cerevisiae, the TAND region of TAF1 displays marked differences in the primary structure relative to Drosophila TAF1 (11% identity) yet possesses transcriptional activity both in vivo and in vitro. Here we present structural and functional studies of yeast TAND1 and TAND2 regions (residues 10–37, and 46–71, respectively). Our NMR data show that, in yeast, TAND1 contains two α-helices (residues 16–23, 30–36) and TAND2 forms a mini β-sheet structure (residues 53–56, 61–64). These TAND1 and TAND2 structured regions interact with the concave and convex sides of the saddle-like structure of TBP, respectively. Present NMR, mutagenesis and genetic data together elucidate that the minimal region (TAND1 core) required for GAL4-dependent transcriptional activation corresponds to the first helix region of TAND1, while the functional core region of TAND2, involved in direct interaction with TBP convex α-helix 2, overlaps with the mini β-sheet region.

Inhibition of host transcription by vesicular stomatitis virus involves a novel mechanism that is independent of phosphorylation of TATA-binding protein (TBP) or association of TBP with TBP-associated factor subunits.

The matrix (M) protein of vesicular stomatitis virus (VSV) is a potent inhibitor in vivo of transcription by all three host RNA polymerases (RNAP). In the case of host RNA polymerase II (RNAPII), the inhibition is due to lack of activity of the TATA-binding protein (TBP), which is a subunit of the basal transcription factor TFIID. Despite the potency of M protein-induced inhibition in vivo, experiments presented here show that M protein cannot directly inactivate TFIID in vitro. Addition of M protein to nuclear extracts from uninfected cells did not inhibit transcription activity, indicating that the inhibition is indirect and is mediated through host factors. The host factors that are known to regulate TBP activity include phosphorylation by host kinases and association with different TBP-associated factor (TAF) subunits. However, TBP in VSV-infected cells was found to be assembled normally with its TAF subunits, as shown by ion exchange high-pressure liquid chromatography and sedimentation velocity analysis. A normal pattern of phosphorylation of TBP in VSV-infected cells was also observed by pH gradient gel electrophoresis. Collectively, these data indicate that M protein inactivates TBP activity in RNAPII-dependent transcription by a novel mechanism, since the known mechanisms for regulating TBP activity cannot account for the inhibition.

A new regulatory domain on the TATA-binding protein.

Recognition of the TATA box by the TATA-binding protein (TBP) is a highly regulated step in RNA polymerase II-dependent transcription. Several proteins have been proposed to regulate TBP activity, yet the TBP domains responsive to all these regulators have not been defined. Here we describe a new class of TBP mutants that increase transcription from core promoters in vivo. The majority of these mutations alter amino acids that cluster tightly on the TBP surface, defining a new TBP regulatory domain. The mutant TBP proteins are defective for binding the transcriptional regulator yNC2, are resistant to inhibition by yNC2 in vitro and exhibit allele-specific genetic interactions with yNC2. These results provide strong biochemical and genetic evidence that TBP is directly repressed in vivo, and define a new TBP domain important for transcriptional regulation.

Functional interaction of HCMV immediate-early 2 protein and hTAFII130/135

RNA polymerases I, II, and III require the TATA-binding protein (TBP) to initiate promoter-specific transcription. We have separated HeLa TBP into four phosphocellulose fractions that elicit polymerase specificity in supplying TBP activity to TBP-depleted pol II and pol III transcription reactions. Polymerase specificity might arise in part through distinct TBP-associated factors (TAFs), which have recently been identified in pol I and II transcription. However, the requirement for pol III TAFs has not been established. Here we show that classical pol III transcription involves a minimum of two novel TAFs: TAF-172 and TAF-L. Not only does TAF-172 activate pol III transcription, but it also inhibits the binding of TBP to the TATA box, thereby repressing pol II transcription. The TBP-TAF-172-TAF-L complex can replace TFIIIB both in transcription reactions reconstituted with TFIIIC and in template commitment assays. Thus SL1, TFIID, and TFIIIB might be functionally similar TBP-TAF complexes that direct pol I, II, and III transcription, respectively.

Phosphorylation of yeast TBP by protein kinase CK2 reduces its specific binding to DNA

Protein kinase CK2 is a ubiquitous Ser/Thr kinase which phosphorylates a large number of proteins including several transcription factors. Recombinant Xenopus laevis CK2 phosphorylates both recombinant Saccharomyces cerevisiae and Schizosaccharomyces pombe TATA binding protein (TBP). The phosphorylation of TBP by CK2 reduces its binding activity to the TATA box. CK2 copurifies with the transcription factor IID (TFIID) complex from HeLa cell extracts and phosphorylates several of the TBP-associated factors within TFIID. Taken together these findings argue for a role of CK2 in the control of transcription by RNA polymerase II through the modulation of the binding activity of TBP to the TATA box.

A cis-acting element 7 bp upstream of the ESF-1-binding motif is involved in E1A 13S autoregulation of the adenovirus 12 TS2 promoter.

Transcription of the E1A gene of the highly oncogenic adenovirus 12 (Ad12) initiates at two start sites (TS1 and TS2). We have previously shown that the E2F and ATF motifs distal of TS1 co-operatively participate in E1A autostimulation from the TS1 promoter region. Here we report the identification of a second E2F-like target region (E2DFII) immediately upstream of the E1A-stimulating factor 1 binding site (ESF-1), important for 13S-mediated autoactivation from TS2. Reporter constructs lacking distinct TS2 cis-acting elements were analysed for their levels of CAT expression in the absence and presence of the E1A 13S protein in transient expression assays. In the absence of 13S, full promoter activity was observed only for a construct containing all elements (the E2F-like motif, and E-Box and the TATA element). Promoter activation increased significantly in Ad12 E1A-co-transfected cells. Induction by the 13S protein was also detected for the construct containing a non-functional ESF-1 sequence. Our results indicate that the E2F-like motif is responsible for activation medicated by the 13S protein from TS2, while ESF-1-or TATA-binding protein activity were not involved. Additionally, the TATA sequence appeared to be dispensable for transactivation. Gel-shift experiments using the E2F-like promoter element as a probe indicated the binding of an E2F-5 or E2F-5-like transcription factor to this region. We conclude that transcription through the TS1 as well as the TS2 promoter region is stimulated by the Ad 12 13S protein. Moreover, transfection of the construct including both TS1 and TS2 indicates an E2F-site-mediated synergism between both regions with respect ot E1A-induced transactivation.

The TATA-binding protein and associated factors are components of pol III transcription factor TFIIIB

RNA polymerases I, II, and III require the TATA-binding protein (TBP) to initiate promoter-specific transcription. We have separated HeLa TBP into four phosphocellulose fractions that elicit polymerase specificity in supplying TBP activity to TBP-depleted pol II and pol III transcription reactions. Polymerase specificity might arise in part through distinct TBP-associated factors (TAFs), which have recently been identified in pol I and II transcription. However, the requirement for pol III TAFs has not been established. Here we show that classical pol III transcription involves a minimum of two novel TAFs: TAF-172 and TAF-L. Not only does TAF-172 activate pol III transcription, but it also inhibits the binding of TBP to the TATA box, thereby repressing pol II transcription. The TBP-TAF-172-TAF-L complex can replace TFIIIB both in transcription reactions reconstituted with TFIIIC and in template commitment assays. Thus SL1, TFIID, and TFIIIB might be functionally similar TBP-TAF complexes that direct pol I, II, and III transcription, respectively.