TATA-binding Protein Complex Research Articles

Taf1 N-terminal domain 2 (TAND2) of TFIID promotes formation of stable and mobile unstable TBP-TATA complexes

In eukaryotes, TATA-binding protein (TBP) occupancy of the core promoter globally correlates with transcriptional activity of class II genes. Elucidating how TBP is delivered to the TATA box or TATA-like element is crucial to understand the mechanisms of transcriptional regulation. A previous study demonstrated that the inhibitory DNA binding (IDB) surface of human TBP plays an indispensable role during the two-step formation of the TBP-TATA complex, first assuming an unstable and unbent intermediate conformation, and subsequently converting slowly to a stable and bent conformation. The DNA binding property of TBP is altered by physical contact of this surface with TBP regulators. In the present study, we examined whether the interaction between Taf1 N-terminal domain 2 (TAND2) and the IDB surface affected DNA binding property of yeast TBP by exploiting TAND2-fused TBP derivatives. TAND2 promoted formation of two distinct types of TBP-TATA complexes, which we arbitrarily designated as complex I and II. While complex I was stable and similar to the well-characterized original TBP-TATA complex, complex II was unstable and moved along DNA. Removal of TAND2 from TBP after complex formation revealed that continuous contact of TAND2 with the IDB surface was required for formation of complex II but not complex I. Further, TFIIA could be incorporated into the complex of TAND2-fused TBP and the TATA box, which was dependent on the amino-terminal non-conserved region of TBP, implying that this region could facilitate the exchange between TAND2 and TFIIA on the IDB surface. Collectively, these findings provide novel insights into the mechanism by which TBP is relieved from the interaction with TAND to bind the TATA box or TATA-like element within promoter-bound TFIID.

Promoters of genes encoding β-amylase, albumin and globulin in food plants have weaker affinity for TATA-binding protein as compared to non-food plants: in silico analysis.

It is generally accepted that during the domestication of food plants, selection was focused on their productivity, the ease of their technological processing into food, and resistance to pathogens and environmental stressors. Besides, the palatability of plant foods and their health benefits could also be subjected to selection by humans in the past. Nonetheless, it is unclear whether in antiquity, aside from positive selection for beneficial properties of plants, humans simultaneously selected against such detrimental properties as allergenicity. This topic is becoming increasingly relevant as the allergization of the population grows, being a major challenge for modern medicine. That is why intensive research by breeders is already underway for creating hypoallergenic forms of food plants. Accordingly, in this paper, albumin, globulin, and β-amylase of common wheat Triticum aestivum L. (1753) are analyzed, which have been identified earlier as targets for attacks by human class E immunoglobulins. At the genomic level, we wanted to find signs of past negative selection against the allergenicity of these three proteins (albumin, globulin, and β-amylase) during the domestication of ancestral forms of modern food plants. We focused the search on the TATA-binding protein (TBP)-binding site because it is located within a narrow region (between positions -70 and -20 relative to the corresponding transcription start sites), is the most conserved, necessary for primary transcription initiation, and is the best-studied regulatory genomic signal in eukaryotes. Our previous studies presented our publicly available Web service Plant_SNP_TATA_Z-tester, which makes it possible to estimate the equilibrium dissociation constant (KD) of TBP complexes with plant proximal promoters (as output data) using 90 bp of their DNA sequences (as input data). In this work, by means of this bioinformatics tool, 363 gene promoter DNA sequences representing 43 plant species were analyzed. It was found that compared with non-food plants, food plants are characterized by significantly weaker affinity of TBP for proximal promoters of their genes homologous to the genes of common-wheat globulin, albumin, and β-amylase (food allergens) (p < 0.01, Fisher's Z-test). This evidence suggests that in the past humans carried out selective breeding to reduce the expression of food plant genes encoding these allergenic proteins.

Identification of appropriate reference genes for qPCR analyses of placental expression of SLC7A3 and induction of SLC5A1 in porcine endometrium

IntroductionEndometria and placentae undergo developmental changes that affect the stability of genes used as references for normalization of qPCR data. We identified genes that are stable within the porcine endometrium and placenta throughout pregnancy, and elucidated the temporal/spatial mRNA localization of the glucose and arginine transporters, solute carrier family (SLC) 5A1 and SLC7A3, respectively. Materials and MethodsqPCR was performed for 10 genes within porcine endometria from Days 5, 11, and 15 of the estrous cycle and 11, 15, 25, 40, 60, and 85 of pregnancy; and chorioallantois from Days 30, 35, 45, 50, 60, and 85. Gene stability was analyzed using GeNorm and NormFinder algorithms. qPCR and in situ hybridization determined temporal/spatial localization of SLC5A1 and SLC7A3 at the uterine-placental interface. ResultsThe geometric mean of TATA-binding protein (TBP), hypoxanthine phosphoribosyl transferase 1 (HPRT1), and tubulin alpha 1B (TUBA1B) provides acceptable reference values for porcine placenta. The geometric mean of TBP, beta actin (ACTB), and succinate dehydrogenase complex subunit A flavoprotein (SDHA) is acceptable for endometria. SLC5A1 is induced by estrogen in endometrial luminal epithelium (LE) on Days 12 and 13 of pregnancy. SLC7A3 is expressed in the chorion. Discussion and ConclusionUsing appropriate reference genes resulted in complementary results between qPCR and in situ hybridization techniques for SLC5A1 and SLC7A3 mRNAs. SLC5A1 is induced in uterine LE by estrogen of trophectoderm origin when the blastocyst is free-floating and dependent on glucose from the endometrium, and SLC7A3 is expressed by the established placenta to support fetal growth.

Prediction and verification of the influence of the rs367781716 SNP on the interaction of the ТАТА-binding protein with the promoter of the human АВСА9 gene

The high-throughput sequencing project 1000 Genomes made it possible to catalog and utilize genetic loci and single nucleotide polymorphisms (SNPs) in medicine. The analysis of SNP markers allows physicians to optimize treatment. However, tens of millions of unannotated SNPs correspond to a gigantic number of false positive (false negative) candidate SNP markers that are selected by computer methods for comparing their frequency in patients with that in healthy people. This approach contributes to the undervaluation of clinically relevant SNPs and to unnecessary computational expenses for the verification of neutral SNPs. The preclinical empirical verification of possible candidate SNP markers may eliminate neutral SNPs from the dataset. In the present study, using the SNP_TATA_Comparator web service, we found the unannotated SNP rs367781716: the substitution of ancestral T (health) with a minor C at position–37 before the transcription initiation site of the ABCA9 gene. This SNP significantly reduces the affinity of TATA-binding protein (TBP) for this gene’s promoter and corresponds to the deficiency (low protein level) of the ABCA9 gene product (the transporter ATP-binding cassette A9) in patients with the–37C allele. For preclinical empirical verification of rs367781716, we used an electrophoretic mobility shift assay (EMSA) to measure the rates of association (ka) and dissociation (kd) of the complexes of TBP with an oligonucleotide matching either allele–37C or–37T of the ABCA9 gene. We found that the rate of association (ka) of the TBP/TATA complex for the minor allele is lower by a factor of 2.4 than that for the ancestral allele. We calculated the empirical value of the change in the equilibrium constant of dissociation (KD = kd/ka), which characterizes the binding affinity of TBP for a promoter containing the TATA box. This empirical value matched the value predicted by the SNP_TATA_Comparator within the allowable margin of error of the measurements and calculations. We also determined the half-life and Gibbs free energy of the complex of TBP with the ABCA9 promoter. Possible phenotypic manifestations of the candidate SNP marker rs367781716 are discussed.

A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation.

TFIID-a complex of TATA-binding protein (TBP) and TBP-associated factors (TAFs)-is a central component of the Pol II promoter recognition apparatus. Recent studies have revealed significant downregulation of TFIID subunits in terminally differentiated myocytes, hepatocytes and adipocytes. Here, we report that TBP protein levels are tightly regulated by the ubiquitin-proteasome system. Using an in vitro ubiquitination assay coupled with biochemical fractionation, we identified Huwe1 as an E3 ligase targeting TBP for K48-linked ubiquitination and proteasome-mediated degradation. Upregulation of Huwe1 expression during myogenesis induces TBP degradation and myotube differentiation. We found that Huwe1 activity on TBP is antagonized by the deubiquitinase USP10, which protects TBP from degradation. Thus, modulating the levels of both Huwe1 and USP10 appears to fine-tune the requisite degradation of TBP during myogenesis. Together, our study unmasks a previously unknown interplay between an E3 ligase and a deubiquitinating enzyme regulating TBP levels during cellular differentiation.

Quantitative mass spectrometry of TATA binding protein-containing complexes and subunit phosphorylations during the cell cycle

BackgroundProgression through the cell cycle is accompanied by tightly controlled regulation of transcription. On one hand, a subset of genes is expressed in a cell cycle-dependent manner. On the other hand, a general inhibition of transcription occurs during mitosis.Genetic and genome-wide studies suggest cell cycle regulation at the level of transcription initiation by protein complexes containing the common DNA-binding subunit TATA binding protein (TBP). TBP is a key player in regulating transcription by all three nuclear RNA polymerases. It forms at least four distinct protein complexes with TBP-associated factors (TAFs): SL1, B-TFIID, TFIID, and TFIIIB. Some TAFs are known to remain associated with TBP during the cell cycle. Here we analyze all TAFs and their phosphorylation status during the cell cycle using a quantitative mass spectrometry approach.ResultsTBP protein complexes present in human cells at the G2/M and G1/S transitions were analyzed by combining affinity purification with quantitative mass spectrometry using stable isotope labeling with amino acids in cell culture (SILAC). Phosphorylations were mapped and quantified after enrichment of tryptic peptides by titanium dioxide. This revealed that subunit stoichiometries of TBP complexes remained intact, but their relative abundances in nuclear extracts changed during the cell cycle. Several novel phosphorylations were detected on subunits of the TBP complexes TFIID and SL1. G2/M-specific phosphorylations were detected on TAF1, TAF4, TAF7, and TAFI41/TAF1D, and G1/S-specific dephosphorylations were detected on TAF3. Many phosphorylated residues were evolutionary conserved from human to zebrafish and/or drosophila, and were present in conserved regions suggesting important regulatory functions.ConclusionsThis study provides the first quantitative proteomic analysis of human TBP containing protein complexes at the G2/M and G1/S transitions, and identifies new cell cycle-dependent phosphorylations on TAFs present in their protein complex. We speculate that phosphorylation of complex-specific subunits may be involved in regulating the activities of TBP protein complexes during the cell cycle.

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.

Quantitative Proteomics Reveals Regulation of Dynamic Components within TATA-binding Protein (TBP) Transcription Complexes

Affinity purification in combination with isotope labeling of proteins has proven to be a powerful method to discriminate specific from nonspecific interactors. However, in the standard SILAC (stable isotope labeling by amino acids in cell culture) approach dynamic components may easily be assigned as nonspecific. We compared two affinity purification protocols, which in combination revealed information on the dynamics of protein complexes. We focused on the central component in eukaryotic transcription, the human TATA-binding protein, which is involved in different complexes. All known TATA-binding protein-associated factors (TAFs) were detected as specific interactors. Interestingly one of them, BTAF1, exchanged significantly in cell extracts during the affinity purification. The other TAFs did not display this behavior. Cell cycle synchronization showed that BTAF1 exchange was regulated during mitosis. The combination of the two affinity purification protocols allows a quantitative approach to identify transient components in any protein complex.

Presence of the anti-leukemic nucleotide analog, 2-chloro-2′-deoxyadenosine-5′-monophosphate, in a promoter sequence alters DNA binding of TATA-binding protein (TBP)

2-Chlorodeoxyadenosine (CldAdo, Cladribine), a nucleoside analog used in the treatment of hairy cell leukemia, is phosphorylated and incorporated into DNA, but is not an absolute chain terminator. We hypothesized that the presence of a chlorine molecule projecting into the DNA minor groove would affect DNA:protein-binding interactions. Here, we investigated recognition of and binding to double-stranded CldAMP-substituted TATA promoter sequences by human TATA-binding protein (TBP) using mobility shift assays. Depending on the site, CldAMP in place of dAMP within a TATA sequence decreased in vitro TBP binding by ∼30% to 55% compared to control sites. When bound to a CldAMP-substituted TATA box, however, the TBP complex was more resistant to polyanions, suggesting enhanced stability. Limited exposure of the TBP:DNA complex to proteases indicated that TBP conformation was altered on CldAMP-substituted DNA compared to control. Further, binding of transcription factor IIB to TBP was diminished on analog-containing TATA sequences. These results suggest normal TBP-binding interactions—specifically recognition, stability, and conformation—are disrupted by CldAMP insertion into eukaryotic promoter sequences.

Non-optimal TATA Elements Exhibit Diverse Mechanistic Consequences

To reveal mechanistic differences in transcription initiation between variant TATA elements, in vivo and in vitro assays of the functional activity of 14 different sequences were compared. Variant elements exhibited particular degrees of activation in vivo but universally were unable to support the -fold activation observed for an element consisting of TATAAA. Each element was classified by its functional activity for in vitro interaction with TATA-binding protein (TBP), TFIIA, and TFIIB. Certain off-consensus TATA elements form poor binding sites for TBP and this compromised interaction interferes with higher order complex formation with TFIIA and/or TFIIB. Other elements are only modestly decreased for TBP binding but dramatically affected for higher order complex formation. Another distinct category is comprised of two elements (CATAAA and TATAAG), which are not affected in the initial formation of the TBP, TFIIA-TBP, or TFIIB-TBP complexes. However, CATAAA and TATAAG are unable to form a stable TFIIA-TBP-DNA complex in vitro. Moreover, fusion of TFIIA to TBP specifically restores activity from these two elements in vivo. Taken together, these results indicate that the interplay between the sequence of the TATA element and the components of the general transcription machinery can lead to variations in the formation of functional complexes and/or the stability of these complexes. These differences offer distinct opportunities for an organism to exploit diverse steps in the regulation of gene expression depending on the precise TATA element sequence at a given gene.

Crystal structure of TBP‐interacting protein (Tk‐TIP26) and implications for its inhibition mechanism of the interaction between TBP and TATA‐DNA

TATA-binding protein (TBP)-interacting protein from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1 (Tk-TIP26) is a possible transcription regulatory protein in Thermococcales. Here, we report the crystal structure of Tk-TIP26 determined at 2.3 A resolution with multiple-wavelength anomalous dispersion (MAD) method. The overall structure of Tk-TIP26 consists of two domains. The N-terminal domain forms an alpha/beta structure, in which three alpha-helices enclose the central beta-sheet. The topology of this domain is similar to that of holliday junction resolvase Hjc from Pyrococcus furiosus. The C-terminal domain comprises three alpha-helices, six beta-strands, and two 3(10)-helices. In the dimer structure of Tk-TIP26, two molecules are related with the crystallographic twofold axis, and these molecules rigidly interact with each other via hydrogen bonds. The complex of Tk-TIP26/Tk-TBP is isolated and analyzed by SDS-PAGE and gel filtration column chromatography, resulting in a stoichiometric ratio of the interaction between Tk-TIP26 and Tk-TBP of 4:2, i.e., two dimer molecules of Tk-TIP26 formed a complex with one dimeric TBP. The electrostatic surfaces of Tk-TIP26 and TBP from Pyrocuccus woesei (PwTBP) allowed us to build a model of the Tk-TIP26/TBP complex, and to propose the inhibition mechanism where two dimer molecules of Tk-TIP26 bind to a dimeric TBP, preventing its binding to TATA-DNA.

Mot1-mediated control of transcription complex assembly and activity

Mot1 is an essential Snf2/Swi2-related ATPase and TATA-binding protein (TBP)-associated factor (TAF). In vitro, Mot1 utilizes ATP hydrolysis to disrupt TBP-DNA complexes, but the relationship of this activity to Mot1's in vivo function is unclear. Chromatin immunoprecipitation was used to determine how Mot1 affects the assembly of preinitiation complexes (PICs) at Mot1-controlled promoters in vivo. We find that the Mot1-repressed HSP26 and INO1 promoters are both regulated by TBP recruitment; inactivation of Mot1 leads to increased PIC formation coincident with derepression of transcription. For the Mot1-activated genes BNA1 and URA1, inactivation of Mot1 also leads, remarkably, to increased TBP binding to the promoters, despite the fact that transcription of these genes is obliterated in mot1 cells. In contrast, levels of Taf1, TFIIB, and RNA polymerase II are reduced at Mot1-activated promoters in mot1 cells. These results suggest that Mot1-mediated displacement of TBP underlies its mechanism of repression and activation at these genes. We suggest that at activated promoters, Mot1 disassembles transcriptionally inactive TBP, thereby facilitating the formation of a TBP complex that supports functional PIC assembly.

Efficient Binding of NC2·TATA-binding Protein to DNA in the Absence of TATA

Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters. This prevents the assembly of transcription factor (TF) IIA and TFIIB and leads to repression of RNA polymerase II transcription. Here we have revisited the interactions of NC2.TBP with DNA. We show that NC2.TBP complexes exhibit a significantly reduced preference for TATA box sequences compared with TBP and TBP.TFIIA complexes. In chromatin immunoprecipitations, NC2 is found on a variety of human TATA-containing and TATA-less promoters. Substantial amounts of NC2 are present in a complex with TBP in bulk chromatin. A complex of NC2.TBP displays a K(D) for DNA of approximately 2 x 10(-9) m for a 35-bp major late promoter oligonucleotide. While preferentially recognizing promoter-bound TBP, NC2 also accelerates TBP binding to promoters and stabilizes TBP.DNA complexes. Our data suggest that NC2 controls TBP binding and maintenance on DNA that is largely independent of a canonical TATA sequence.

Structure and Function of the TFIID Complex

This chapter focuses on the structure and function of transcription factor IID (TFIID) complex and proposes several functions of TFIID in transcription regulation. These roles include promoter recognition, co-activator function, and several enzymatic activities. The purification of TFIID was soon followed by the identification of its subunits. The amino acid sequences of many TATA-binding protein (TBP)-associated factors (TAFs) revealed a high level of conservation. TFIID structure has changed little through evolution, with all species containing at least 13 conserved subunits. A consensus on the nomenclature of TAFs has been developed to avoid confusion and is used throughout the chapter. Structural studies of TBP and TAF sub complexes provided insights into the mechanisms of TFIID function and complex organization. A low resolution structure of the whole TFIID complex has also been obtained using electron microscopy. The complex has a horseshoe shape, consisting of 3–4 lobes connected by flexible regions. It is suggested that the concave face may represent the DNA binding surface and that DNA might be accommodated in the central channel. TBP was mapped to the central lobe, which is also where transcription factor IIA and transcription factor IIB bind. Further determination of individual subunit positions will be essential for the understanding of how TFIID functions as a complex.

Mot1 associates with transcriptionally active promoters and inhibits association of NC2 in Saccharomyces cerevisiae.

Mot1 stably associates with the TATA-binding protein (TBP), and it can dissociate TBP from DNA in an ATP-dependent manner. Mot1 acts as a negative regulator of TBP function in vitro, but genome-wide transcriptional profiling suggests that Mot1 positively affects about 10% of yeast genes and negatively affects about 5%. Unexpectedly, Mot1 associates with active RNA polymerase (Pol) II and III promoters, and it is rapidly recruited in response to activator proteins. At Pol II promoters, Mot1 association requires TBP and is strongly correlated with the level of TBP occupancy. However, the Mot1/TBP occupancy ratio at both Mot1-stimulated and Mot1-inhibited promoters is high relative to that at typical promoters, strongly suggesting that Mot1 directly affects transcriptional activity in a positive or negative manner, depending on the gene. The effect of Mot1 at the HIS3 promoter region depends on the functional quality and DNA sequence of the TATA element. Unlike TBP, Mot1 association is largely independent of the Srb4 component of Pol II holoenzyme, and it also can occur downstream of the promoter region. Mot1 removes TBP, but not TBP complexes or preinitiation complexes, from inappropriate genomic locations. Mot1 inhibits the association of NC2 with promoters, suggesting that the TBP-Mot1 and TBP-NC2 complexes compete for promoter occupancy in vivo. We speculate that Mot1 does not form transcriptionally active TBP complexes but rather regulates transcription in vivo by modulating the activity of free TBP and/or by affecting promoter DNA structure.

Kinetic Studies of the TATA-binding Protein Interaction with Cisplatin-modified DNA

The TATA-binding protein (TBP) recognizes the TATA box element of transcriptional promoters and recruits other initiation factors. This essential protein binds selectively to cisplatin-damaged DNA. Electrophoretic mobility shift assays were performed to study the kinetics of TBP binding both to the TATA box and to cisplatin-damaged DNA in different sequence contexts. TBP binds with high affinity (K(d) = 0.3 nm) to DNA containing site-specific cisplatin 1,2-intrastrand d(GpG) cross-links. The k(on) and k(off) values for the formation of these TBP complexes are 1-3 x 10(5) m(-1) s(-1) and approximately 1-5 x 10(-4) s(-1), respectively, similar to the corresponding values for the formation of a TBP-TATA box complex. In electrophoretic mobility shift assay competition assays, cisplatin-damaged DNA extensively sequesters TBP from its natural binding site, the TATA box. Nine DNA probes were prepared to determine the flanking sequence dependence of TBP binding to cisplatin-modified DNA. TBP clearly displays sequence context selectivity for platinated DNA, very similar to but not as dramatic as that of the high mobility group protein HMGB1. When TBP was added to an in vitro nucleotide excision repair assay, it specifically shielded cisplatin-modified 1,2-(GpG) intrastrand cross-links from repair. These results indicate that TBP is likely to be a key protein in mediating the cytotoxicity of cisplatin.

Biochemical Characterization of the TATA-binding Protein-Gal4 Activation Domain Complex

It has been suggested that complexes between gene-specific activators and the TATA-binding protein (TBP) play an important role in the expression of many genes. However, few detailed studies of well defined activator-TBP complexes have been reported. An analysis of the biochemical properties of the complex formed by the acidic activation domain (AAD) of the yeast activator Gal4 and TBP is presented here. This is shown to be composed of two AAD and one TBP molecule. DNA binding experiments reveal that TATA-containing DNAs and the Gal4 AAD bind TBP competitively, suggesting that the AAD and TATA boxes recognize overlapping surfaces of TBP. The kinetics of the formation and dissociation of the AAD(2)-TBP complex is also probed. The impact of these findings on models for Gal4-mediated transcriptional activation is considered.

TAF-Containing and TAF-independent forms of transcriptionally active TBP in vivo.

Transcriptional activity in yeast strongly correlates with promoter occupancy by general factors such as TATA binding protein (TBP), TFIIA, and TFIIB, but not with occupancy by TBP-associated factors (TAFs). Thus, TBP exists in at least two transcriptionally active forms in vivo. The TAF-containing form corresponds to the TFIID complex, whereas the form lacking TAFs corresponds to TBP itself or to some other TBP complex. Heat shock treatment altered the relative utilization of these TBP forms, with TFIID being favored. Promoter-specific variations in the association of these distinct forms of TBP may explain why only some yeast genes require TFIID for transcriptional activity in vivo.

Nuclear import of the TATA-binding protein: mediation by the karyopherin Kap114p and a possible mechanism for intranuclear targeting.

Binding of the TATA-binding protein (TBP) to the promoter is the first and rate limiting step in the formation of transcriptional complexes. We show here that nuclear import of TBP is mediated by a new karyopherin (Kap) (importin) family member, Kap114p. Kap114p is localized to the cytoplasm and nucleus. A complex of Kap114p and TBP was detected in the cytosol and could be reconstituted using recombinant proteins, suggesting that the interaction was direct. Deletion of the KAP114 gene led to specific mislocalization of TBP to the cytoplasm. We also describe two other potential minor import pathways for TBP. Consistent with other Kaps, the dissociation of TBP from Kap114p is dependent on RanGTP. However, we could show that double stranded, TATA-containing DNA stimulates this RanGTP-mediated dissociation of TBP, and is necessary at lower RanGTP concentrations. This suggests a mechanism where, once in the nucleus, TBP is preferentially released from Kap114p at the promoter of genes to be transcribed. In this fashion Kap114p may play a role in the intranuclear targeting of TBP.

Interaction of the transcription factor TFIID with simian virus 40 (SV40) large T antigen interferes with replication of SV40 DNA in vitro.

Simian virus 40 (SV40) large tumor (T) antigen is the major regulatory protein that directs the course of viral infection, primarily by interacting with host cell proteins and modulating their functions. Initiation of viral DNA replication requires specific interactions of T antigen bound to the viral origin of DNA replication with cellular replication proteins. Transcription factors are thought to stimulate initiation of viral DNA replication, but the mechanism of stimulation is poorly understood. Since the transcription factor TATA-binding protein (TBP) binds to sequences within the origin of replication and interacts specifically with T antigen, we examined whether TBP complexes stimulate SV40 DNA replication in vitro. On the contrary, we found that depletion of TBP complexes from human cell extracts increased their ability to support viral DNA replication, and readdition of TBP complexes to the depleted extracts diminished their activity. We have mapped the sites of interaction between the proteins to residues 181 to 205 of T antigen and 184 to 220 of TBP. Titration of fusion proteins containing either of these peptides into undepleted cell extracts stimulated their replication activity, suggesting that they prevented the T antigen-TBP interaction that interfered with replication activity. TBP complexes also interfered with origin DNA unwinding by purified T antigen, and addition of either the T antigen or the TBP fusion peptide relieved the inhibition. These results suggest that TBP complexes associate with a T-antigen surface that is also required for origin DNA unwinding and viral DNA replication. We speculate that competition among cellular proteins for T antigen may play a role in regulating the course of viral infection.