Yeast TATA Box Binding Protein Research Articles

Structural convergence endows nuclear transport receptor Kap114p with a transcriptional repressor function toward TATA-binding protein

The transcription factor TATA-box binding protein (TBP) modulates gene expression in nuclei. This process requires the involvement of nuclear transport receptors, collectively termed karyopherin-β (Kap-β) in yeast, and various regulatory factors. In previous studies we showed that Kap114p, a Kap-β that mediates nuclear import of yeast TBP (yTBP), modulates yTBP-dependent transcription. However, how Kap114p associates with yTBP to exert its multifaceted functions has remained elusive. Here, we employ single-particle cryo-electron microscopy to determine the structure of Kap114p in complex with the core domain of yTBP (yTBPC). Remarkably, Kap114p wraps around the yTBPC N-terminal lobe, revealing a structure resembling transcriptional regulators in complex with TBP, suggesting convergent evolution of the two protein groups for a common function. We further demonstrate that Kap114p sequesters yTBP away from promoters, preventing a collapse of yTBP dynamics required for yeast responses to environmental stress. Hence, we demonstrate that nuclear transport receptors represent critical elements of the transcriptional regulatory network.

Isolation of cDNAs encoding for two distinct isoforms of the TATA-Box binding proteins from common bean (Phaseolus vulgaris L.)

The TATA-box binding protein (TBP) is one of the 4 DNA-binding proteins that has been shown to associate with the proximal promoter region (−295) of the gene for bean seed storage protein phaseolin. The −295 promoter is essential for spatial and temporal control of the phaseolin gene expression. We designed a pair of degenerated primers based on the highly conserved sequence of the carboxyl-terminal domain of yeast TBP and used PCR to amplify the corresponding sequence from the bean cDNA. By using the amplified fragment as a probe, we screened a cDNA library derived from poly A(+) RNA from developing bean seeds and isolated 2 nearly full-length cDNA clones (813 and 826 bp long). The cDNAs encode 2 distinct isoforms of bean TBP, PV1 and PV2, each with an open reading frame of 200 amino acid residues. The 2 cDNA sequences share an 85.8% overall nucleotide sequence identity, with the coding region showing a higher degree of identity (94.4%) than the 5′- and 3′-untranslated regions (69%). The deduced amino acid sequence of the bean TBP isoforms differ in only 3 amino acid residues at positions 5, 9, and 16, all located in the amino-terminal region. The carboxyl-terminal domain of 180 amino acid residues shows a high degree (>82%) of evolutionary sequence conservation with the TBP sequences from other eukaryotic species. This domain possesses the 3 highly conserved structural motifs, namely the 2 direct repeat sequences, a central basic region rich in basic amino acid residues, and a region similar to the sigma factor of prokaryote. On the basis of this and other findings, we suggest that higher plants in general may have at least 2 copies of TBP gene, presumably resulting from the global duplication of the genome.

Gal4-VP16 and Gal4-AH increase the orientational and axial specificity of TATA box recognition by TATA box binding protein.

Previous work has shown that binding of the TATA box binding protein (TBP) to the TATA box is a rate-limiting step during pre-initiation complex (PIC) formation. Although the transcription of eukaryotic genes normally proceeds in one direction, studies in solution have shown that TBP lacks the information necessary to orient itself on the TATA box. Instead, yeast TBP binds TATA-containing promoters in two orientations that are related by a 180 rotation about TBP's pseudo-2-fold symmetry axis. Recruitment of PIC components by gene-specific activators is considered a primary mechanism of transcriptional enhancement. Here we ask whether activators might function, at least in part, by increasing the fraction of PICs assembled with TBP bound in the orientation necessary for transcription. We use DNA affinity cleavage and a TBP-phenanthroline-copper conjugate to monitor the orientation of TBP in the presence of the well-studied activators Gal4-VP16 and Gal4-AH. In the absence of a transcriptional activator, only 51% of the TBP x TATA box complexes were bound in the orientation necessary for the initiation of transcription. However, in the presence of saturating Gal4-VP16, 87% of the TBP bound to the TATA box was oriented correctly at equilibrium. This increase in orientational specificity corresponds to a free energy difference (Delta Delta G(obs)) of 1.1 kcal x mol(-1) and was accompanied by a dramatic increase in axial specificity, reminiscent of the effects of transcription factors TFIIB and TFIIA reported previously. Gal4-AH also enhanced the orientational and axial specificity of the TBP x TATA complex, although to a lesser extent. We suggest that these effects on specificity represent a variation of recruitment, since they require direct interactions between the activator and a PIC component but only increase the effective concentration of the correctly oriented PIC component. These findings add to increasing evidence that recruitment may encompass a broad range of mechanisms.

Promoter-specific activation defects by a novel yeast TBP mutant compromised for TFIIB interaction

TFIIB is an RNA polymerase II general transcription factor (GTF) that has also been implicated in the mechanism of action of certain promoter-specific activators (see, for examples, [1–11]). TFIIB enters the preinitiation complex (PIC) primarily through contact with the TATA box binding protein (TBP), an interaction mediated by three TBP residues [12–14]. To study the role of TFIIB in transcription activation in vivo, we randomly mutagenized these three residues in yeast TBP and screened for promoter-specific activation mutants. One mutant bearing a single conservative substitution, TBP-E186D, is the focus of this study. As expected, TBP-E186D binds normally to the TATA box but fails to support the entry of TFIIB into the PIC. Cells expressing TBP-E186D are viable but have a severe slow-growth phenotype. Whole-genome expression analysis indicates that transcription of 17% of yeast genes are compromised by this mutation. Chimeric promoter analysis indicates that the region of the gene that confers sensitivity to the TBP-E186D mutation is the UAS (upstream activating sequence), which contains the activator binding sites. Most interestingly, other TBP mutants that interfere with different interactions (TFIIB, TFIIA, or the TATA box) and a TFIIB mutant defective for interaction with TBP all manifest distinct and selective promoter-specific activation defects. Our results implicate the entry of TFIIB into the PIC as a critical step in the activation of certain promoters and reveal diverse mechanisms of transcription activation.

High Affinity Interaction of Yeast Transcriptional Regulator, Mot1, with TATA Box-binding Protein (TBP)

Yeast Mot1, an essential ATP-dependent regulator of basal transcription, removes TATA box-binding protein (TBP) from TATA sites in vitro. Complexes of Mot1 and Spt15 (yeast TBP), radiolabeled in vitro, were immunoprecipitated with anti-TBP (or anti-Mot1) antibodies in the absence of DNA, showing Mot1 binds TBP in solution. Mot1 N-terminal deletions (residues 25-801) abolished TBP binding, whereas C-terminal ATPase domain deletions (residues 802-1867) did not. Complex formation was prevented above 200 mm salt, consistent with electrostatic interaction. Correspondingly, TBP variants lacking solvent-exposed positive charge did not bind Mot1, whereas a mutant lacking positive charge within the DNA-binding groove bound Mot1. ATPase-defective mutant, Mot1(D1408N), which inhibits growth when overexpressed (but is suppressed by co-overexpression of TBP), bound TBP normally in vitro, suggesting it forms nonrecyclable complexes. N-terminal deletions of Mot1(D1408N) were not growth-inhibitory. C-terminal deletions were toxic when overexpressed, and toxicity was ameliorated by TBP co-overproduction. Residues 1-800 of Mot1 are therefore necessary and sufficient for TBP binding. The N terminus of 89B, a tissue-specific Drosophila Mot1 homolog, bound the TBP-like factor, dTRF1. Native Mot1 and derivatives deleterious to growth localized in the nucleus, whereas nontoxic derivatives localized to the cytosol, suggesting TBP binding and nuclear transport of Mot1 are coupled.

Purification and Enzymic Properties of Mot1 ATPase, a Regulator of Basal Transcription in the Yeast Saccharomyces cerevisiae

The 1867-residue Mot1 protein is a member of a superfamily of ATPases, some of which are helicases, that interact with protein-nucleic acid assemblies. Mot1 is an essential regulator of RNA polymerase II-dependent transcription in vivo and dissociates TATA box-binding protein (TBP)-DNA complexes in vitro. Mot1-(His)(6) was purified to apparent homogeneity from yeast extracts. The preparation efficiently dissociated TBP.TATA complexes, suggesting that no other protein or cofactor is required. Mot1 behaved as a non-globular monomer in hydrodynamic studies, and no association was detected between differentially tagged co-expressed Mot1 constructs. ATPase activity was stimulated about 10-fold by high ionic strength or alkaline pH, or by deletion of the N-terminal TBP-binding segment, suggesting that the N-terminal domain negatively regulates the C-terminal ATPase domain (Mot1C). Correspondingly, at moderate salt concentration, Mot1 ATPase (but not Mot1C) was stimulated >/=10-fold by yeast TBP, suggesting that interaction with TBP relieves a conformational constraint in Mot1. Double- or single-stranded TATA-containing DNA did not affect ATPase activity of Mot1 or Mot1C, with or without TBP. Mot1 did not exhibit detectable helicase activity in strand displacement assays using substrates with flush ends or 5'- or 3'-overhangs. Mot1-catalyzed dissociation of TBP from DNA was not prevented by a psoralen cross-link positioned immediately preceding the TATA sequence. Thus, Mot1 most likely promotes release of TBP from TATA-containing DNA by causing a structural change in TBP itself, rather than by strand unwinding.

Regulation of Gene Expression by Multiple Forms of TFIID and Other Novel TAFII-Containing Complexes

Ear ly work employed in vitro t ranscr ipt ion react ions to character ize protein factors required for accura te t ranscr ipt ion by RNA polymerase II (Pol II). Chromatographica lly separable protein fract ions were tested for their ability to produce cor rect ly in it ia ted t ranscr ipt s from promoter DNA fragments [1–3]. These differen t chromatographic fract ions, which conta in dist inct act ivit ies, were shown to be required for basa l Pol II t ranscr ipt ion and designa ted genera l Pol II t ranscr ipt ion factors, TFIIs. After the in it ia l character iza t ion of these fract ions it was shown by using differen t footpr in t ing techniques tha t a factor presen t in the TFIID fract ions preferen t ia lly binds to the TATA box, a core element shared by many promoters. Subsequent ly it was shown tha t in it ia t ion of the t ranscr ipt ion by Pol II from a promoter region requires the sequence-specific binding of the TFIID complex to the promoter . Binding of TFIID is followed by the subsequent ordered in teract ions of the other basa l factors (TFIIA, TFIIB, TFIIE, TFIIF , TFIIH, and TFIIJ ) and Pol II to yield a product ive prein it ia t ion complex (PIC) [4 – 6]. In tense effor t s to pur ify the cor responding protein or protein complex(es) in the differen t mammalian TFIID act ivity-conta in ing fract ions to homogeneity were unsuccessfu l. A breakthrough came with the discovery tha t a simila r TATA box binding act ivity exist s in the yeast S accharom yces cerevisiae [7], bu t tha t th is act ivity could be isola ted as a single polypept ide of 27 kDa [8]. This observa t ion set the stage for clon ing of yeast TATA box binding protein (TBP) [9 –11]. The cloning of the gene encoding yeast TBP allowed the isola t ion of severa l TBP-encoding genes from other eukaryotes [12, 13]. The compar ison of the size of recombinant TBP with tha t of mammalian TFIID fract ions subsequent ly revea led tha t TFIID, or a t least a fract ion of the TFIID act ivity, is a t igh t ly associa ted protein complex composed of TBP and a number (10 –13) of TBP-associa ted factors (TAF IIs) [13–16]. The discovery tha t TBP is not only a component of the TFIID complex, but is a lso present in t ranscr ipt ion complexes funct ion ing in Pol I and III t ranscr ipt ion , placed TBP as a cent ra l player in t ranscr ipt ion [12, 17]. The fact tha t recombinant TBP can replace the huge mult iprotein complex, TFIID, in basa l Pol II t ranscr ipt ion react ions in vitro, but tha t in metazoan cells TBP does not seem to exist a lone and is a lways associa ted with mult iprotein complexes, st imula ted much in terest in the role of TAF IIs in t ranscr ipt ion regula t ion . Today most of the TFIID components from S . cerevisiae, Drosophila, and humans have been ident ified (Table 1) and par t ia lly character ized [16, 18 –20]. Here we review the roles of TFIID and other novel TAF II-conta in ing complexes in the regula t ion of class II gene expression . We emphasize in par t icu la r those findings tha t under line the rela t ively recent idea tha t many TAF II-conta in ing complexes, and not a single TFIID, play impor tan t roles in t ranscr ipt iona l regu la t ion .

Polymerase (Pol) III TATA box-binding protein (TBP)-associated factor Brf binds to a surface on TBP also required for activated Pol II transcription.

The TATA box-binding protein (TBP) plays an essential role in transcription by all three eukaryotic nuclear RNA polymerases, polymerases (Pol) I, II, and III. In each case, TBP interacts with class-specific TBP-associated factors (TAFs) to form class-specific transcription initiation factors. For yeast Pol III transcription, TBP associates with Brf (from TFIIB-related factor) and B", two Pol III TAFs, to form Pol III transcription factor TFIIIB. Here, we identify TBP surface residues that are required for interaction with yeast Pol III TAFs. Ninety-one human TBP surface residue mutants with radical substitutions were analyzed for the ability to form stable gel shift complexes with purified Brf and B" and for their activities for in vitro synthesis of yeast U6 snRNA. Mutations in a large positively charged epitope extending from the top (that is, on the surface opposite the DNA-facing "saddle" of TBP) and onto the side of the first TBP repeat inhibited binding to Brf (residues K181, L185, R186, E206, R231, L232, R235, K236, R239, Q242, K243, K249, and F250). A triple-mutant TBP (R231E + R235E + R239S) had greatly reduced activity for yeast U6 snRNA gene transcription while remaining active for Pol II basal transcription. Similar results were observed when selected mutations were introduced into yeast TBP at equivalent positions. A C-terminal fragment of Brf lacking the region of homology with TFIIB retains the ability to bind TBP-DNA complexes (G. Kassavetis, C. Bardeleben, A. Kumar, E. Ramirez, and E. P. Geiduschek, Mol. Cell. Biol. 17:5299-5306, 1997); the same TBP mutations reduced binding by this fragment. Mutations in TBP residues that interact with TFIIB did not affect Brf binding or U6 gene transcription. These results indicate that Brf and TFIIB interact differently with TBP. An extensively overlapping epitope on the top surface of TBP was found previously to be required for activated Pol II transcription and has been hypothesized to interact with Pol II TAFs. Our results map the surface of TBP that interacts with Brf and suggest that Pol II and Pol III TAFs interact with the same surface of TBP.

Bidirectional binding of the TATA box binding protein to the TATA box.

By selective attachment of a DNA cleavage agent to specific residues in the yeast TATA box binding protein (yTBP), we demonstrate that, in solution, yTBP binds to the TATA boxes of both the adenovirus major late promoter and the yeast CYC1 promoter with only a modest preference in orientation and binds well to several overlapping binding sites. The general factors TFIIA and TFIIB each increase the rotational and translational selectivity of yTBP but are not sufficient, at least individually, to confer a unique polarity to the preinitiation complex. We conclude that TBP alone cannot define the productive orientation of general factor assembly on a promoter.

Design of TATA box-binding protein/zinc finger fusions for targeted regulation of gene expression.

Fusing the TATA box-binding protein (TBP) to other DNA-binding domains may provide a powerful way of targeting TBP to particular promoters. To explore this possibility, a structure-based design strategy was used to construct a fusion protein, TBP/ZF, in which the three zinc fingers of Zif268 were linked to the COOH terminus of yeast TBP. Gel shift experiments revealed that this fusion protein formed an extraordinarily stable complex when bound to the appropriate composite DNA site (half-life up to 630 h). In vitro transcription experiments and transient cotransfection assays revealed that TBP/ZF could act as a site-specific repressor. Because the DNA-binding specificities of zinc finger domains can be systematically altered by phage display, it may be possible to target such TBP/zinc finger fusions to desired promoters and thus specifically regulate expression of endogenous genes.

The Crystal Structure of a Hyperthermophilic Archaeal TATA-box Binding Protein

This study analyzes the three-dimensional structure of the TATA-box binding protein (TBP) from the hyperthermophilic archaea Pyrococcus woesei. The crystal structure of P. woeseiTBP ( PwTBP) was solved at 2.2 Å by X-ray diffraction and as expected from sequence homology (36% to 41% identical to eukaryotic TBPs) its overall structure is very similar to eukaryotic TBPs. The thermal unfolding transition temperature of this protein was measured by differential scanning calorimetry to be 101°C, which is more than 40°C higher than that of yeast TBP. Preliminary titration calorimetry data show that the affinity of PwTBP for its DNA target, unlike its eukaryotic counterparts, is enhanced by increasing the temperature and salt concentration. The structure reveals possible explanations for this thermostability and these unusual DNA binding properties. The crystal structure of this hyperthermostable protein was compared to its mesophilic homologs and analyzed for differences in the native structure that may contribute to thermostability. Differences found were: (1) a disulfide bond not found in mesophilic counterparts; (2) an increased number of surface electrostatic interactions; (3) more compact protein packing. The presumed DNA binding surface of PwTBP, like its eukaryotic counterparts, is hydrophobic but the electrostatic profile surrounding the protein is relatively neutral compared to the asymmetric positive potential that surrounds eukaryotic TBPs. The total reliance on a hydrophobic interface with DNA may explain the enhanced affinity of PwTBP for its DNA promoter at higher temperatures and increased salt concentration.

TATA-box DNA binding activity and subunit composition for RNA polymerase III transcription factor IIIB from Xenopus laevis.

The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding protein (TBP) and polymerase III-specific TBP-associated factors (TAFs). Previous studies have shown that DNA oligonucleotides containing the consensus TATA-box sequence inhibit polymerase III transcription, implying that the DNA binding domain of TBP is exposed in TFIIIB. We have investigated the TATA-box DNA binding activity of Xenopus TFIIIB, using transcription inhibition assays and a gel mobility shift assay. Gel shift competition assays with mutant and nonspecific DNAs demonstrate the specificity of the TFIIIB-TATA box DNA complex. The apparent dissociation constant for this protein-DNA interaction is approximately 0.4 nM, similar to the affinity of yeast TBP for the same sequence. TFIIIB transcriptional activity and TATA-box binding activity cofractionate during a series of four ion-exchange chromatographic steps, and reconstituted transcription reactions demonstrate that the TATA-box DNA-protein complex contains TFIIIB TAF activity. Polypeptides with apparent molecular masses of 75 and 92 kDa are associated with TBP in this complex. These polypeptides were renatured after elution from sodium dodecyl sulfate-gels and tested individually and in combination for TFIIIB TAF activity. Recombinant TBP along with protein fractions containing the 75- and 92-kDa polypeptides were sufficient to reconstitute TFIIIB transcriptional activity and DNA binding activity, suggesting that Xenopus TFIIIB is composed of TBP along with these polypeptides.

Yeast RSP5 and its human homolog hRPF1 potentiate hormone-dependent activation of transcription by human progesterone and glucocorticoid receptors.

We have developed a system in Saccharomyces cerevisiae in which agonist-dependent transcriptional activity of the human progesterone receptor (hPR) is elevated to the point that it compromises cell growth. Screens for suppressors of this phenotype led to the demonstration that RSP5 is involved in hPR transactivation. Expression of RSP5 in yeast cells potentiated hPR and human glucocorticoid receptor (hGR) transcriptional activity and increased the efficacy of weak agonists of these receptors. Remarkably, expression of this yeast protein in mammalian cells had a similar effect on PR and GR transcriptional activity. Importantly, a human homolog of RSP5, hRPF1, functioned identically in mammalian cells. Previously, it has been demonstrated that RSP5 overexpression in yeast cells suppressed mutations within SPT3, a protein which interacts with the TATA-box-binding protein (TBP), suggesting that RSP5 and SPT3 operate in the same regulatory pathway. In support of this observation, we have shown that SPT3 enhances the activity of RSP5 on GR and PR when tested in yeast or mammalian cells. We conclude from these experiments that the regulatory pathways in which RSP5 and SPT3 operate in yeast cells are conserved in higher eukaryotes. Additionally, since SPT3 has been shown to contact yeast TBP directly and is the likely homolog of human TBP-associated factor TAFII18, we propose that RSP5/hRPF1 and SPT3 establish a functional link between activated PR and GR and the general transcription apparatus.

The Neuroendocrine Protein 7B2 Contains Unusually Potent Transcriptional Activating Sequences

The expression of the 7B2 protein, secreted from a variety of neural and endocrine tissues, increases dramatically in specific neuroendocrine tumors. We have recently shown that human 7B2 can act as a molecular chaperone in the deaggregation of proteins in vitro. In order to identify polypeptides which might bind 7B2 in vivo, the yeast two-hybrid system was employed. Surprisingly, mere covalent linkage of 7B2 to the DNA-binding domains of two yeast transcription activators, Ace1 and Gal4, activates transcription from the ACE1 and GAL4 operon. 7B2′s ability to activate nuclear transcription surpasses that of Ace1 and compares favourably with the strong activation domain of the tumor suppressor protein, p53. Our results suggest that 7B2 must possess an activating sequence, a domain which defines all transcriptional activator proteins. Like the acidic activation domains of some transpirational activators, 7B2 also binds the yeast TATA-box binding protein, an essential polypeptide in the basic transcription machinery. Deletion analysis of the gene encoding 7B2 reveals two independent transcriptional activating sequences in the 185 amino acid protein. It is therefore conceivable that 7B2 not only has a functional role in the secretory pathway but also in the nucleus. Moreover, these findings raise an intriguing question regarding the activation domains of 7B2 and their possible link to 7B2′s oncogenic potential.

Recombinant TBP, transcription factor IIB, and RAP30 are sufficient for promoter recognition by mammalian RNA polymerase II.

Initiation of transcription by RNA polymerase II is a complex, multistep process which requires several accessory factors in addition to the polymerase itself. A critical event in transcription initiation is the specific association of RNA polymerase II with promoter DNA. In this report we show that three eukaryotic polypeptides, produced in Escherichia coli and purified to near homogeneity, constitute a minimal set of general transcription factors both necessary and sufficient for specific and stable promoter binding by RNA polymerase II. These polypeptides are the yeast TATA box binding protein TBP, the human general initiation factor TFIIB, and human RAP30, the small subunit of RAP30/74 (or transcription factor IIF). Formation of the polymerase-containing complex required only the TATA box, and not the initiator element (Inr), of the adenovirus major late promoter which was used in these experiments.