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When targeted to sequences adjacent to a TATA element, pyrrole-imidazole (Py-Im) polyamides inhibit the DNA binding activity of TATA box binding protein (TBP) and basal transcription by RNA polymerase II. In the present study, we scanned the human immunodeficiency virus type 1 promoter for polyamide inhibition of TBP binding and transcription using a series of DNA constructs in which a polyamide binding site was placed at various distances from the TATA box. Polyamide interference with either TBP-DNA or TFIID-TFIIA-DNA contacts both upstream and downstream of the TATA element resulted in inhibition of transcription. Our results define important protein-DNA interactions outside of the TATA element and suggest that transcription inhibition of selected gene promoters can be achieved with polyamides that target unique sequences within these promoters at a distance from the TATA element. Our studies also demonstrate the utility of the Py-Im polyamides for discovery of functionally important protein-DNA contacts involved in transcription.

Interleukin-2 (IL-2) is a cytokine critical for the proper stimulation of T-cells during the mammalian immune response. Shortly after T-cell stimulation, transcription of the IL-2 gene is upregulated. Here, we studied the kinetic mechanism of basal transcription at the IL-2 promoter using a human in vitro RNA polymerase II transcription system. We experimentally divided the transcription reaction into discrete steps, including preinitiation complex formation, initiation, escape commitment, and promoter escape. Using pre-steady state approaches, we measured the rate at which each of these steps occurs. We found that the rate of functional preinitiation complex formation limits the overall rate of transcription at the IL-2 promoter under the conditions described here. Furthermore, we found that the recruitment of TFIIF and RNA polymerase II to a TFIID/TFIIA/TFIIB/promoter complex dictates the rate of preinitiation complex formation. The rate of synthesis of 28 nt RNA from preinitiation complexes was rapid compared to the rate of preinitiation complex formation. Moreover, we found that the synthesis of a four nucleotide RNA was necessary and sufficient to rapidly complete the escape commitment step of transcription at the IL-2 promoter. Comparative experiments with the adenovirus major late promoter revealed that, while the overall mechanism of transcription is the same at the two promoters, promoter sequence and/or architecture dictate the rate of promoter escape. We present a kinetic model for a single round of basal transcription at the IL-2 promoter that provides insight into mechanisms by which the IL-2 gene is transcriptionally regulated., (Copyright 2001 Academic Press.)

A topological model for transcription initiation by RNA polymerase II (RNAPII) has recently been proposed. This model stipulates that wrapping of the promoter DNA around RNAPII and the general initiation factors TBP, TFIIB, TFIIE, TFIIF and TFIIH induces a torsional strain in the DNA double helix that facilitates strand separation and open complex formation. In this report, we show that TFIIA, a factor previously shown to both stimulate basal transcription and have co-activator functions, is located near the cross-point of the DNA loop where it can interact with TBP, TFIIE56, TFIIE34, and the RNAPII-associated protein (RAP) 74. In addition, we demonstrate that TFIIA can stimulate basal transcription by stimulating the functions of both TFIIE34 and RAP74 during the initiation step of the transcription reaction. These results provide novel insights into mechanisms of TFIIA function.

Human transcription factor TFIID contains the TATA-binding protein (TBP) and several TBP-associated factors (TAF(II)s). To elucidate the structural organization and function of TFIID, we expressed and characterized the product of a cloned cDNA encoding human TAF(II)135 (hTAF(II)135). Comparative far Western blots have shown that hTAF(II)135 interacts strongly with hTAF(II)20, moderately with hTAF(II)150, and weakly with hTAF(II)43 and hTAF(II)250. Consistent with these observations and with sequence relationships of hTAF(II)20 and hTAF(II)135 to histones H2B and H2A, respectively, TFIID preparations that contain higher levels of hTAF(II)135 also contain higher levels of hTAF(II)20, and the interaction between hTAF(II)20 and hTAF(II)135 is critical for human TFIID assembly in vitro. From a functional standpoint, hTAF(II)135 has been found to interact strongly and directly with hTFIIA and (within a complex that also contains hTBP and hTAF(II)250) to specifically cooperate with TFIIA to relieve TAF(II)250-mediated repression of TBP binding and function on core promoters. Finally, we report a functional synergism between TAF(II)s and the TRAP/Mediator complex in activated transcription, manifested as hTAF(II)-mediated inhibition of basal transcription and a consequent TRAP requirement for both a high absolute level of activated transcription and a high and more physiological activated/basal transcription ratio. These results suggest a dynamic TFIID structure in which the switch from a basal hTAF(II)-enhanced repression state to an activator-mediated activated state on a promoter may be mediated in part through activator or coactivator interactions with hTAF(II)135.

DNA sequences that determine transcriptional regulation of a typical eukaryotic gene consist of a core promoter, which serves as a binding site for the GTF TFIID, and regulatory promoter or enhancer sequences, which bind transcriptional activators. The RNA polymerase II transcription machinery consists of over 50 proteins which are thought to bind to the core promoter in as few as two steps: binding of TFIIA-TFIID, followed by binding of a large pre-assembled holoenzyme complex consisting of the remaining GTFs, RNA polymerase II and associated regulatory proteins. Activators function to increase binding of the transcription machinery to the promoter in at least two ways: (i) simple protein-protein interactions with activators increases the affinity of the transcription machinery for the promoter, and (ii) some activators stabilize a conformation of the TFIIA-TFIID-DNA complex that enhances binding of the holoenzyme. Recent studies have identified many co-activators that function with activators to increase transcription by the RNA polymerase II transcription machinery. Although some co-activators may serve as bridges to connect activators with the transcription machinery, the mechanism of action of many co-activators has not yet been determined.

High levels of gene transcription by RNA polymerase II depend on high rates of transcription initiation and reinitiation. Initiation requires recruitment of the complete transcription machinery to a promoter, a process facilitated by activators and chromatin remodelling factors. Reinitiation probably occurs through a different pathway. After initiation, a subset of the transcription machinery remains at the promoter, forming a platform for assembly of a second transcription complex. Here we describe the isolation of a reinitiation intermediate that includes transcription factors TFIID, TFIIA, TFIIH, TFIIE and Mediator. This intermediate can act as a scaffold for formation of a functional reinitiation complex. Formation of this scaffold is dependent on ATP and TFIIH. The scaffold is stabilized in the presence of the activator Gal4-VP16, but not Gal4-AH, suggesting a new role for some activators and Mediator in promoting high levels of transcription.

The compartmentalization of DNA in the nucleus of eukaryotic cells establishes a connection between the nuclear transport machinery and the transcriptional apparatus. General transcription factors, as well as specific transcriptional activators and repressors, such as p53 and NF-AT, need to be imported into the nucleus following their translation. In addition, nuclear transport plays a crucial role in regulating the activity of many transcription factors.

A variant polyadenylation signal, which is conserved and employed by mammalian hepadnaviruses, has a sequence resembling that of the TATA box. We report here that this composite box manifests all the promoter characteristics. It binds effectively TATA-binding protein with TFIIB and TFIIA in a synergistic manner. This capacity, however, is lost when the box is converted to a canonical and simple poly(A) signal. Furthermore, we show that it has promoter activity and supports transcription of reporter genes preferentially in liver-derived cells, a characteristic behavior of the hepatitis B virus (HBV) promoters. In addition, we show that the HBV noncanonical poly(A) signal supports transcription initiation from the viral genome, suggesting that it is a genuine promoter, possibly of the polymerase/reverse transcriptase gene. Finally, we found that this deviant poly(A) signal is crucial for HBV replication since a viral mutant with a canonical poly(A) box is impaired in replication. Our data, therefore, raise the interesting and novel possibility that a composite poly(A) box might have a dual function. At the level of DNA it functions as a promoter to initiate transcription, whereas at the level of RNA it serves as a poly(A) signal to process RNA. An interesting outcome of this strategy of gene expression is that it provides a novel mechanism for the synthesis of an approximately genome length transcript.

Transcription of eukaryotic structural genes requires the assembly of RNA polymerase II and the general transcription factors (GTFs) on the promoter to form a pre-initiation complex (PIC). Among these, TFIID is the major sequence-specific DNA-binding component; the other GTFs enter the PIC primarily through protein-protein interactions. TFIID is composed of the TATA-box-binding protein (TBP) and multiple TBP-associated factors (TAFIIs). Unexpectedly, TAFIIs have also been found in other multi-subunit complexes involved in transcription. Whereas TBP is a general transcription factor, a variety of in vivo studies have demonstrated that TAFIIs are highly promoter selective. Here I review studies on the role of TAFIIs in genome-wide transcription and their mechanism of action.

The TATA binding protein (TBP) plays a central role in eukaryotic and archael transcription initiation. We describe the isolation of a novel 23-kDa human protein that displays 41% identity to TBP and is expressed in most human tissue. Recombinant TBP-related protein (TRP) displayed barely detectable binding to consensus TATA box sequences but bound with slightly higher affinities to nonconsensus TATA sequences. TRP did not substitute for TBP in transcription reactions in vitro. However, addition of TRP potently inhibited basal and activated transcription from multiple promoters in vitro and in vivo. General transcription factors TFIIA and TFIIB bound glutathione S-transferase-TRP in solution but failed to stimulate TRP binding to DNA. Preincubation of TRP with TFIIA inhibited TBP-TFIIA-DNA complex formation and addition of TFIIA overcame TRP-mediated transcription repression. TRP transcriptional repression activity was specifically reduced by mutations in TRP that disrupt the TFIIA binding surface but not by mutations that disrupt the TFIIB or DNA binding surface of TRP. These results suggest that TFIIA is a primary target of TRP transcription inhibition and that TRP may modulate transcription by a novel mechanism involving the partial mimicry of TBP functions.

Adenovirus E1B 55,000-molecular-weight protein (55K) binds to host cell p53, stabilizing it, greatly increasing its affinity for its cognate DNA-binding site, and converting it from a regulated activator to a constitutive repressor. Here we analyzed the mechanism of repression by the p53-E1B 55K complex. E1B 55K repression requires that 55K be tethered to the promoter by binding directly to DNA-bound p53. Transcription from an assembled, p53-activated preinitiation complex was not repressed by the subsequent addition of E1B 55K, suggesting that either sites of 55K interaction with p53 or targets of 55K in the preinitiation complex are blocked. Specific E1B 55K repression was observed in reactions lacking TFIIA and with recombinant TATA-binding protein in place of TFIID, conditions under which p53 does not activate transcription. Thus, E1B 55K does not simply inhibit a p53-specific activation mechanism but rather blocks basal transcription. As a consequence, E1B 55K may repress transcription from any promoter with an associated p53-binding site, no matter what other activators associate with the promoter. E1B 55K did not repress basal transcription in reactions with recombinant and highly purified general transcription factors and RNA polymerase II but rather required a corepressor that copurifies with the polymerase.

By in situ chromosomal hybridization, and by somatic cell and radiation hybrid analysis, we have determined the genomic position of the human genes encoding four TAFII subunits of TFIID (TAFII150, TAFII105, TAFII68, TAFII18), the three subunits of TFIIA (TFIIA35 and TFIIA19, both encoded by the same gene, and TFIIA12), CDK8, and SURB7. All of these proteins are bona fide components of human class II holoenzymes as well as targets of signal transduction pathways that regulate genome expression. The genes encoding them are present in the human genome in a single copy and are localized at 8q23, 18q11.2, 17q11.1-11.2, 1p21, 14q31, 15q21-23, 13q12, and 12p12, respectively. We have mapped all of them to chromosomal regions where hereditary genetic diseases have been localized or which are involved in malignancies, which makes them potential candidates for a causal involvement in these phenotypes.

Two coordinate forms of transcriptional synergy mediate eukaryotic gene regulation: the greater-than-additive transcriptional response to multiple promoter-bound activators, and the sigmoidal response to increasing activator concentration. The mechanism underlying the sigmoidal response has not been elucidated but is almost certainly founded on the cooperative binding of activators and the general machinery to DNA. Here we explore that mechanism by using highly purified transcription factor preparations and a strong Epstein-Barr virus promoter, BHLF-1, regulated by the virally encoded activator ZEBRA. We demonstrate that two layers of cooperative binding govern transcription complex assembly. First, the architectural proteins HMG-1 and -2 mediate cooperative formation of an enhanceosome containing ZEBRA and cellular Sp1. This enhanceosome then recruits transcription factor IIA (TFIIA) and TFIID to the promoter to form the DA complex. The DA complex, however, stimulates assembly of the enhanceosome itself such that the entire reaction can occur in a highly concerted manner. The data reveal the importance of reciprocal cooperative interactions among activators and the general machinery in eukaryotic gene regulation.

Posttranslational modification of general transcription factors may be an important mechanism for global gene regulation. The general transcription factor IIA (TFIIA) binds to the TATA binding protein (TBP) and is essential for high-level transcription mediated by various activators. Modulation of the TFIIA-TBP interaction is a likely target of transcriptional regulation. We report here that Toa1, the large subunit of yeast TFIIA, is phosphorylated in vivo and that this phosphorylation stabilizes the TFIIA-TBP-DNA complex and is required for high-level transcription. Alanine substitution of serine residues 220, 225, and 232 completely eliminated in vivo phosphorylation of Toa1, although no single amino acid substitution of these serine residues eliminated phosphorylation in vivo. Phosphorylated TFIIA was 30-fold more efficient in forming a stable complex with TBP and TATA DNA. Dephosphorylation of yeast-derived TFIIA reduced DNA binding activity, and recombinant TFIIA could be stimulated by in vitro phosphorylation with casein kinase II. Yeast strains expressing the toa1 S220/225/232A showed reduced high-level transcriptional activity at the URA1, URA3, and HIS3 promoters but were viable. However, S220/225/232A was synthetically lethal when combined with an alanine substitution mutation at W285, which disrupts the TFIIA-TBP interface. Phosphorylation of TFIIA could therefore be an important mechanism of transcription modulation, since it stimulates TFIIA-TBP association, enhances high-level transcription, and contributes to yeast viability.

RNA polymerase II transcribes the mRNA-encoding genes and the majority of the small nuclear RNA (snRNA) genes. The formation of a minimal functional transcription initiation complex on a TATA-box-containing mRNA promoter has been well characterized and involves the ordered assembly of a number of general transcription factors (GTFs), all of which have been either cloned or purified to near homogeneity. In the human RNA polymerase II snRNA promoters, a single element, the proximal sequence element (PSE), is sufficient to direct basal levels of transcription in vitro. The PSE is recognized by the basal transcription complex SNAPc. SNAPc, which is not required for transcription from mRNA-type RNA polymerase II promoters such as the adenovirus type 2 major late (Ad2ML) promoter, is thought to recruit TATA binding protein (TBP) and nucleate the assembly of the snRNA transcription initiation complex, but little is known about which GTFs other than TBP are required. Here we show that the GTFs IIA, IIB, IIF, and IIE are required for efficient RNA polymerase II transcription from snRNA promoters. Thus, although the factors that recognize the core elements of RNA polymerase II mRNA and snRNA-type promoters differ, they mediate the recruitment of many common GTFs.

The retinoblastoma tumor suppressor protein (pRB) is a transcriptional repressor, critical for normal cell cycle progression. We have undertaken studies using a highly purified reconstituted in vitro transcription system to demonstrate how pRB can repress transcriptional activation mediated by the E2F transcription factor. Remarkably, E2F activation became resistant to pRB-mediated repression after the establishment of a partial (TFIIA/TFIID) preinitiation complex (PIC). DNase I footprinting studies suggest that E2F recruits TFIID to the promoter in a step that also requires TFIIA and confirm that recruitment of the PIC by E2F is blocked by pRB. These studies suggest a detailed mechanism by which E2F activates and pRB represses transcription without the requirement of histone-modifying enzymes.

Potential pathways that could account for observed rapid rates of transcription reinitiation were explored. A nuclear extract system was established in which reinitiation rates were observed to be kinetically facilitated and in which the rate was sensitive to TATA box mutation. Kinetic facilitation of functional complex formation could be mimicked by pre-assembling activator and certain general transcription factors on the promoter and then adding nuclear extract. The minimal activated complex with this characteristic contained general factors TFIID and TFIIA. The ability of the TFIID:TFIIA complex to complete assembly rapidly was reduced by the same TATA box mutation that reduced reinitiation rate. Band shift experiments also showed that this same mutation lowered the stability of the TBP:TFIIA complex on the DNA. The results suggest that TATA-dependent variations in retention of the TFIID:TFIIA complex after release of the polymerase could be a primary determinant of reinitiation rate, allowing diversity in promoter strength to be related to diversity in TATA element sequences.

We explore the role of histone H1 as a DNA sequence-dependent architectural determinant of chromatin structure and of transcriptional activity in chromatin. The Xenopus laevis oocyte- and somatic-type 5S rRNA genes are differentially transcribed in embryonic chromosomes in vivo depending on the incorporation of somatic histone H1 into chromatin. We establish that this effect can be reconstructed at the level of a single nucleosome. H1 selectively represses oocyte-type 5S rRNA genes by directing the stable positioning of a nucleosome such that transcription factors cannot bind to the gene. This effect does not occur on the somatic-type genes. Histone H1 binds to the 5' end of the nucleosome core on the somatic 5S rRNA gene, leaving key regulatory elements in the promoter accessible, while histone H1 binds to the 3' end of the nucleosome core on the oocyte 5S rRNA genes, specifically blocking access to a key promoter element (the C box). TFIIIA can bind to the somatic 5S rRNA gene assembled into a nucleosome in the presence of H1. Because H1 binds with equivalent affinities to nucleosomes containing either gene, we establish that it is the sequence-selective assembly of a specific repressive chromatin structure on the oocyte 5S rRNA genes that accounts for differential transcriptional repression. Thus, general components of chromatin can determine the assembly of specific regulatory nucleoprotein complexes.

The Xenopus TFIIIA gene is transcribed very efficiently in oocytes. In addition to a TATA element at -30, we show that from -425 to +7 the TFIIIA gene contains only two positive cis elements centered at -267 (element 1) and -230 (element 2). This arrangement of the cis elements in the TFIIIA gene is striking because these two elements are positioned very close to each other yet separated from the TATA element by approximately 190 nucleotides. We show that the 190-nucleotide spacing between the TATA element and the upstream cis elements (elements 1 and 2) is critical for efficient transcription of the gene in oocytes and that a nucleosome is positioned in this intervening region. This nucleosome may act positively on TFIIIA transcription in oocytes by placing transcription factors bound at elements 1 and 2 in a favorable position relative to the transcription complex at the TATA element.

RBP is a cellular protein that functions as a transcriptional repressor in mammalian cells. RBP has elicited great interest lately because of its established roles in regulating gene expression, in Drosophila and mouse development, and as a component of the Notch signal transduction pathway. This report focuses on the mechanism by which RBP represses transcription and thereby regulates expression of a relatively simple, but natural, promoter. The results show that, irrespective of the close proximity between RBP and other transcription factors bound to the promoter, RBP does not occlude binding by these other transcription factors. Instead, RBP interacts with two transcriptional coactivators: dTAFII110, a subunit of TFIID, and TFIIA to repress transcription. The domain of dTAFII110 targeted by RBP is the same domain that interacts with TFIIA, but is disparate from the domain that interacts with Sp1. Repression can be thwarted when stable transcription preinitiation complexes are formed before RBP addition, suggesting that RBP interaction with TFIIA and TFIID perturbs optimal interactions between these coactivators. Consistent with this, interaction between RBP and TFIIA precludes interaction with dTAFII110. This is the first report of a repressor specifically targeting these two coactivators to subvert activated transcription.

The TFIID complex interacts with at least three types of core promoter elements within protein-coding genes, including TATA, initiator (Inr), and downstream promoter elements. We have begun to explore the mechanism by which the TFIID-Inr interaction leads to functional synergy between TATA and Inr elements during both basal and activated transcription. In DNase I footprinting assays, GAL4-VP16 recruited TFIID-TFIIA to core promoters containing either a TATA box, an Inr, or both TATA and Inr elements, with synergistic interactions apparent on the TATA-Inr promoter. Appropriate spacing between the two elements was essential for the synergistic binding. Despite the sequence-specific TFIID-Inr interactions, gel shift experiments revealed that TFIID alone possesses similar affinities for the TATA-Inr and TATA promoters. Interestingly, however, recombinant TFIIA strongly and selectively enhanced TFIID binding to the TATA-Inr promoter, with little effect on binding to the TATA promoter. Studies of the natural adenovirus major late promoter confirmed these findings, despite the existence of specific but nonfunctional TFIID interactions downstream of the Inr in that promoter. These results suggest that a TFIIA-induced conformational change is essential for the sequence-specific TFIID-Inr interaction to occur with sufficient affinity to support the functional synergism between TATA and Inr elements.

Eukaryotic cells are thought to contain a single TATA-binding protein (TBP) that directs transcription by cellular RNA polymerases. Here we report a cell type-specific TBP-related factor (TRF) that can form a stable TRF/IIA/IIB TATA DNA complex and substitute for TBP in directing RNA polymerase II transcription in vitro. Transfection studies reveal that TRF can differentially mediate activation by some enhancer proteins but not others. Like TBP, TRF forms a stable complex containing multiple novel subunits, nTAFs. Antibody staining of embryos and polytene chromosomes reveals cell type-specific expression and gene-selective properties consistent with the shaker/male sterile phenotype of trf mutants. These findings suggest TRF is a homolog of TBP that functions to direct tissue- and gene-specific transcription.

Human transcription factor IIIC2 interacts with the TFIIIA-5S DNA complex and forms a ternary TFIIIA/IIIC2-5S DNA complex. Formation of this complex does not preclude simultaneous binding of TFIIIC2to the B-box sequence of a second template. This suggests that the domain(s) or subunit(s) required for indirect recognition of the 5S promoter by TFIIIC2 are different from those necessary for direct binding of TFIIIC2 to B-box-containing pol III promoters. Whereas TFIIIC2 is only required for transcription of the 'classical' pol III genes, TFIIIC1 is generally required for transcription of all pol III genes, including that of the U6 gene. The activity of TFIIIC1 strongly enhances specific binding of basal pol III factors TFIIIA, TFIIIC2 and the PSE binding protein (PBP) to their cognate promoter elements and it acts independently of the corresponding termination regions. Moreover, we characterize an activity, TFIIIC0, purified from phosphocellulose fraction C, which shows strong DNase I protection of the termination region of several pol III genes and which is functionally and chromatographically distinct from TFIIIC1 and TFIIIC2.

Transcription initiation on protein-encoding genes represents a major control point for gene expression in eukaryotes, and is mediated by RNA polymerase II and a surprisingly complex array of general initiation factors (TFIIA, -B, -D, -E, -F and -H) that are highly conserved from yeast to man. Elucidation of structural and functional features of these factors on model promoters has revealed insights into biochemical mechanisms and provides a basis for understanding their regulation on diverse promoters by gene- and cell-specific activators.

Four new studies of the assembly of different subsets of the preinitiation complex of RNA polymerase II have provided insight into how transcription factors interact with each other and with DNA. A heterotetramer of TBP-associated factors shows a histone-like fold.

Using a genetic screen, we isolated four TATA-binding protein (TBP) mutants that are specifically defective in vivo for the response to acidic activators. In contrast to previously described activation-defective TBP mutants, these TBP derivatives are not specifically defective for interactions with TATA elements or TFIIA. Three of these derivatives interact normally with a TATA element, TFIIA, TFIIB, or an acidic activation domain; presumably, they affect another protein-protein interaction important for transcriptional activation. The remaining derivative (with F-237 replaced by D) binds a TATA element with wild-type affinity, but the TBP-TATA complex has an altered electrophoretic mobility and interacts poorly with TFIIA and TFIIB; this suggests that the conformation of the TBP-TATA element complex plays a role in transcriptional activation. To determine the step at which the TBP derivatives were unable to activate transcription, we utilized an artificial recruitment assay in which TBP is targeted to the promoter via fusion to the LexA DNA-binding domain. Consistent with previous evidence that acidic activators can increase recruitment of TBP to the promoter in vivo, the activation defect of some of these TBP derivatives can be corrected by artificial recruitment. In contrast, the activation defect of the other TBP derivatives is not bypassed by artificial recruitment. Thus, these TBP mutants define two steps in the process of transcriptional stimulation by acidic activators: efficient recruitment to the TATA element and a postrecruitment interaction with a component(s) of the initiation complex.

The binding of TBP (TFIID) to the TATA box has been considered to direct promoter recognition and pre-initiation complex formation because it is the first event leading to basal transcription by RNA polymerase II. Here, we analyse the binding of yeast TBP to a consensus TATAAA box and two point mutations, TAAAAA (inactive) and TATATA (active). Despite the fact that the TAAAAA sequence does not support transcription in vitro, yeast TBP binds the three sequences showing, in this sense, only a limited sequence specificity. However, the TBP-TAAAAA complex cannot be recognised by other basal transcription factors, in particular by TFIIB. DNase I footprinting patterns of the TBP-TAAAAA complex are different from those observed in functional TBP-TATA box complexes, indicating that, most likely, it is a different spatial arrangement of the TBP-DNA complex that prevents formation of the TFIIB-TBP-TAAAAA complex, also seriously impairing entry of TFIIA to the complex. DNA deformability of the A/T-rich sequences appears to be an important determinant in the formation of a productive TBP-TATA complex. These results indicate that the transcriptional competence of A/T-rich sequences is determined not only by TBP binding, but also by the ability of other basal transcription factors to recognise the preformed TBP-DNA complexes.

The antibiotic distamycin A is a DNA minor groove binding drug (MGB) that recognizes a stretch of at least four ATs. The alkylating benzoyl mustard derivative tallimustine (FCE 24517) has powerful anti-tumor activity. Using the electrophoretic mobility shift assay (EMSA) we determined that both compounds can prevent binding of TBP and, with 10-fold higher concentration, TBP-TFIIA (DA) and TBP-TFIIA-TFIIB (DAB) to a TATA box. Once formed, the DA and DAB complexes are more resistant to MGB challenge. Both drugs can inhibit basal in vitro transcription of a minimal TATA-containing promoter and similar concentrations are necessary for binding and transcriptional inhibition. Tallimustine shows strong selectivity by decreasing only correctly initiated transcripts. Even at high doses (20 microM), however, they cannot disturb a competent pre-initiation complex or Pol II progression. This functional in vitro model will provide a way to investigate the activity of sequence-specific DNA binding drugs with potential anti-viral and anti-tumour activity and to develop novel more selective compounds.

The TATA-binding protein (TBP) plays a key role in transcription initiation. Several negative cofactors (NC1, NC2, and Dr1) are known to interact with TBP in a manner that prevents productive interactions of transcription factors TFIIA and TFIIB with promoter-bound TBP. To gain insights into the regulatory interplay on the surface of TBP, we have employed mutant forms of TBP to identify amino acid residues important for interactions with the negative regulatory cofactor NC2 and the general factor TFIIB. The results show the involvement of distinct domains of TBP in these interactions. Residues (Lys-133, Lys-145, and Lys-151) in the basic repeat region are important for interactions with NC2, as well as with TFIIA (Buratowski, S., and Zhou, H. (1992) Science 255, 1130-1132; Lee, D. K., DeJong, J., Hashimoto, S., Horikoshi, M., and Roeder, R. G. (1992) Mol. Cell. Biol. 12, 5189-5196), whereas a residue (Leu-189) in the second stirrup-like loop spanning S2' and S3' is required for interaction with TFIIB. In addition, we demonstrate that NC2 is identical to the previously cloned negative cofactor Dr1. The implications of these results for TBP structure and function are discussed.

The human general transcription factor TFIIA is one of several factors involved in specific transcription by RNA polymerase II, possibly by regulating the activity of the TATA-binding subunit (TBP) of TFIID. TFIIA purified from HeLa extracts consists of 35-, 19-, and 12-kDa subunits. Here we describe the isolation of a cDNA clone (hTFIIA gamma) encoding the 12-kDa subunit. Using expression constructs derived from hTFIIA gamma and TFIIA alpha/beta (which encodes a 55-kDa precursor to the alpha and beta subunits of natural TFIIA), we have constructed a synthetic TFIIA with a polypeptide composition similar to that of natural TFIIA. The recombinant complex supports the formation of a DNA-TBP-TFIIA complex and mediates both basal and Gal4-VP16-activated transcription by RNA polymerase II in TFIIA-depleted nuclear extracts. In contrast, TFIIA has no effect on tRNA and 5S RNA transcription by RNA polymerase III in this system. We also present evidence that both the p55 and p12 recombinant subunits interact with TBP and that the basic region of TBP is critical for the TFIIA-dependent function of TBP in nuclear extracts.

To probe the structure and function of the Saccharomyces cerevisiae general transcription factor TFIIA, we have systematically mutagenized the genes encoding both subunits and analyzed the effects of the mutations both in vivo and in vitro. We found that the central nonconserved region of the large subunit is not essential for function and likely acts as a spacer between the conserved N- and C-terminal regions. Deletion mutagenesis of the large subunit defined a region which is required for TATA binding protein (TBP) interaction. Alanine scanning mutagenesis defined a cluster of four basic residues which are likely required for interaction with DNA in the TBP-DNA complex. Much of the conserved regions of both subunits is required for subunit association, suggesting that these conserved regions fold into compact domains which extensively interact. In vitro transcription performed with extracts from yeast strains with mutations in either the large or the small TFIIA subunit demonstrated that TFIIA stimulates both basal and activated polymerase II (Pol II) transcription. The TFIIA-depleted extracts have normal Pol I and Pol III transcription activity, showing that TFIIA is a Pol II-specific factor. In vivo depletion of TFIIA activity reduced transcription from four different Pol II promoters. Finally, alanine scanning mutagenesis of TFIIA's small subunit has identified at least one mutation which is defective in transcription but which is not defective in subunit association or binding to TBP or TBP-DNA complexes.

TATA boxes are common structural features of eucaryal class II and archaeal promoters. In addition, a gene encoding a polypeptide with sequence similarity to eucaryal TATA-binding protein (TBP) has recently been detected in Archaea, but its relationship to the archaeal transcription factors A (aTFA) and B (aTFB) was unclear. Here, we demonstrate that yeast and human TBP can substitute for aTFB in a Methanococcus-derived archaeal cell-free transcription system. Template-commitment studies show that eucaryal TBP is stably sequestered at the archaeal promoter and that this interaction is further stabilized in combination with aTFA. Binding studies revealed that recognition of an archaeal promoter by TBP involves specific binding to the TATA box. These findings demonstrate a common function of TBP and aTFB and imply a common evolutionary origin of eucaryal and archaeal transcriptional machinery.

Transcriptional activator proteins stimulate the formation of a preinitiation complex that may be distinct from a basal-level transcription complex in its composition and stability. Components of the general transcription factors that form activator-dependent stable intermediates were determined by the use of Sarkosyl and oligonucleotide challenge experiments. High-level transcriptional activation by the Epstein-Barr virus-encoded Zta protein required an activity in the TFIID fraction that is distinct from the TATA-binding protein (TBP) and the TBP-associated factors. This additional activity copurifies with and is likely to be identical to the previously defined coactivator, USA (M. Meisterernst, A. L. Roy, H. M. Lieu, and R. G. Roeder, Cell 66:981-994, 1991). The formation of a stable preinitiation complex intermediate resistant to Sarkosyl required the preincubation of the promoter DNA with Zta, holo-TFIID (TBP and TBP-associated factors), TFIIB, TFIIA, and the coactivator USA. The formation of a Zta response element-resistant preinitiation complex required the preincubation of promoter DNA with Zta, holo-TFIID, TFIIB, and TFIIA. Agarose gel electrophoretic mobility shift showed that a preformed Zta-holo-TFIID-TFIIA complex was resistant to Sarkosyl and to Zta response element oligonucleotide challenge. DNase I footprinting suggests that only Zta, holo-TFIID, and TFIIA make significant contacts with the promoter DNA. These results provide functional and physical evidence that the Zta transcriptional activator influences at least two distinct steps in preinitiation complex assembly, the formation of the stable holo-TFIID-TFIIA-promoter complex and the subsequent binding of TFIIB and a USA-like coactivator.

The human T-lymphotropic virus type I (HTLV-I) promoter contains the structural features of a typical RNA polymerase II (pol II) template. The promoter contains a TATA box 30 bp upstream of the transcription initiation site and binding sites for several pol II transcription factors, and long poly(A)+ RNA is synthesized from the integrated HTLV-I proviral DNA in vivo. Consistent with these characteristics, HTLV-I transcription activity was reconstituted in vitro by using TATA-binding protein, TFIIA, recombinant TFIIB, TFIIE, and TFIIF, TFIIH, and pol II. Transcription of the HTLV-I promoter in the reconstituted system requires RNA pol II. In HeLa whole cell extracts, however, the HTLV-I long terminal repeat also contains an overlapping transcription unit (OTU). HTLV-I OTU transcription is initiated at the same nucleotide site as the RNA isolated from the HTLV-I-infected cell line MT-2 but was not inhibited by the presence of alpha-amanitin at concentrations which inhibited the adenovirus major late pol II promoter (6 micrograms/ml). HTLV-I transcription was inhibited when higher concentrations of alpha-amanitin (60 micrograms/ml) were used, in the range of a typical pol III promoter (VA-I). Neutralization and depletion experiments with three distinct pol II antibodies demonstrate that RNA pol II is not required for HTLV-I OTU transcription. Antibodies to basal transcription factors TATA-binding protein and TFIIB, but not TFIIIC, inhibited HTLV-I OTU transcription. These observations suggest that the HTLV-I long terminal repeat contains overlapping promoters, a typical pol II promoter and a unique pol III promoter which requires a distinct set of transcription factors.

Activator-dependent transcription in mammalian cells requires upstream stimulatory activity (USA)-derived cofactors in addition to those present in TFIID. A novel positive cofactor (PC4) purified from the human USA fraction effected a marked enhancement (up to 85-fold) of GAL4-AH-dependent transcription in conjunction with TFIID and other general factors. Isolation of a corresponding cDNA identified PC4 as a 127 residue single-stranded DNA-binding protein with serine-rich regions near the N-terminus. Recombinant PC4 was functionally equivalent to native PC4, and both proteins markedly enhanced activation by diverse activation domains fused to the DNA-binding domain of GAL4. Recombinant PC4 interacted independently both with free or DNA-bound VP16 activation domains and with free or DNA-bound TFIIA-TBP complexes (but not with TBP alone). These results indicate that PC4 is a general coactivator that functions cooperatively with TAFs and mediates functional interactions between upstream activators and the general transcriptional machinery.

A basal repressor of class II gene transcription was identified, purified, and found to be identical to nonhistone chromosomal protein HMG2. HMG2 was shown to inhibit basal transcription under conditions in which transcription templates form soluble complexes with HMG2. Order-of-addition experiments clearly revealed that HMG2 acted after assembly of a TBP-TFIIA-promoter complex and before formation of the fourth phosphodiester bond by RNA polymerase II. Subsequently, an activity that efficiently counteracted repression of transcription by HMG2 in both TBP- and TFIID-containing transcription systems was isolated. Several lines of evidence suggested that antirepression was mediated by a TFIIH-associated factor. The antirepressor first coeluted with TFIIH, was depleted from this fraction by antibodies directed against the TFIIH subunit p62, was dependent on either ATP or dATP, and then was inhibited by the ATP analogs AMP-PNP and ATP gamma S. Relief of HMG2-mediated repression as well as basal promoter function of TFIIH may involve a helicase that coelutes with TFIIH and displays similar nucleotide specificities. Taken together, these data suggest novel consequences of chromatin-associated HMG proteins and they provide direct evidence for a role of TFIIH-associated enzymes in ATP-dependent antirepression of nonhistone chromosomal proteins.

Transcription initiation by RNA polymerase II is effected by an ordered series of general factor interactions with core promoter elements (leading to basal activity) and further regulated by gene-specific factors acting from distal elements. Both the general factor TFIID (refs 2,3), including the constituent TBP (TATA-binding polypeptide) and associated factors, and the interacting factor TFIIB (refs 9-11) have been implicated as targets for various activators. Towards an understanding of the basis for activator function, including the multiplicity of TBP interactions, we have now identified mutations in yeast TBP that selectively block activator (GAL4-VP16)-dependent but not basal transcription. We further show an effect of GAL4-VP16 on TFIIB recruitment to early preinitiation complexes, and that recruitment is disrupted by TBP mutations that impair its interactions with VP16 (L114K), TFIIB (L189K) or an unidentified component (K211L). Thus, GAL4-VP16 function seems to involve both direct interactions with TBP and a corresponding induction (or stabilization) of an activation-specific TBP-TFIIB-promoter complex.

The past year has provided new insights into the biochemical mechanism of gene activation. Key discoveries include the finding that TFIIA plays an important regulatory role in transcription complex assembly, the TBP-associated factors are direct targets of at least two classes of activator, and a largely pre-assembled transcription complex has been isolated from yeast cells, challenging the step-wise assembly pathway. This review also presents an update on the argument that TFIIB is the target of VP16 and insights into the energetic role of ATP in RNA polymerase II initiation.

In eukaryotes, the TATA box binding protein (TBP) is an integral component of the transcription initiation complexes of all three classes of nuclear RNA polymerases. In this study we have investigated the role of the N-terminal region of human TBP in transcription initiation from RNA polymerase (Pol) I, II and III promoters by using three monoclonal antibodies (mAbs). Each antibody recognizes a distinct epitope in the N-terminal domain of human TBP. We demonstrate that these antibodies differentially affect transcription from distinct classes of promoters. One antibody, mAb1C2, and a synthetic peptide comprising its epitope selectively inhibited in vitro transcription from TATA-containing, but not from TATA-less promoters, irrespective of whether they were transcribed by Pol II or Pol III. Transcription by Pol I, on the other hand, was not affected. Two other antibodies and their respective epitope peptides did not affect transcription from any of the promoters tested. Order of addition experiments indicate that mAb1C2 did not prevent binding of TBP to the TATA box or the formation of the TBP-TFIIA-TFIIB complex but rather inhibited a subsequent step of preinitiation complex formation. These data suggest that a defined region within the N-terminal domain of human TBP may be involved in specific protein-protein interactions required for the assembly of functional preinitiation complexes on TATA-containing, but not on TATA-less promoters.

The nuclear proto-oncoprotein Myc has been implicated in the control of cell proliferation and differentiation. Myc participates in transcription and belongs to the basic-helix-loop-helix (bHLH) family of regulatory proteins. Here we show that Myc interacts with TFII-I, a transcription initiation factor that activates core promoters through an initiator element (Inr). As previously observed for the bHLH activator USF, Myc was found to interact cooperatively with TFII-I at both Inr and upstream E-box promoter elements. However, in this case Myc interactions with TFII-I at the Inr lead to an inhibition of transcription initiation. This inhibition is selective for a TFII-I-dependent (as opposed to TFIIA-dependent) initiation pathway and correlates with the prevention of complex formation between the TATA-binding protein TBP (TFIID tau), TFII-I and the promoter. TBP probably interacts with Myc, but only slowly. These observations indicate that Myc has the potential to interact physically and functionally with components of the general transcription machinery.

The minimal promoter elements required for initiation by RNA polymerase II include the TATA box and/or an initiator element (Inr) at or near the transcription start site. Studies of the adenovirus major late core promoter (containing both elements) have demonstrated an initiation pathway that involves binding of the transcription factor TFIID (or the derived subunit, the TATA-binding protein TBP (TFIID tau)) to the TATA element, which is facilitated by transcription factor TFIIA, followed by sequential interactions of other general factors. Here we describe a novel pathway that requires an intact Inr and the Inr-binding factor TFII-I (ref. 3). Sequential addition of the general factors generated TFII-I-dependent preinitiation complexes different from those formed with TFIIA. Furthermore, TBP bound cooperatively (with only TFII-I) to an Inr-containing TATA-less promoter, suggesting a means for activation of TATA-less promoters, which nonetheless require TFIID (refs 9-11). These observations provide support for functionally distinct pathways which could be subject to differential regulation by specific activators or repressors.

Immunoglobulin heavy chain (IgH) gene transcription in vitro can be reconstituted with a minimal reaction containing only TATA-binding protein (TBP), TFIIB, and RNA polymerase II (pol II) when the template is negatively supercoiled. Transcription from linear DNA templates containing either the IgH or the adenovirus major late promoters (MLPs) requires in addition TFIIF, TFIIE, TFIIH, and a fraction containing TFIIA and TFIIJ. Promoters vary in their activities in the minimal reaction. Initiation at the adenovirus MLP site was not observed in this reaction, even with templates containing negative superhelical density. When only TBP, TFIIB, and pol II were present in the reaction, the more negatively supercoiled the IgH template DNA was, the more active the transcription. It is suggested that the free energy of supercoiling promotes the formation of an open complex for initiation of transcription by the minimal set of transcription factors.

It has previously been shown that excess wild type (wt) p53 can repress the transcriptional activity of a variety of promoters in intact cells. To determine whether this transcriptional repression represented a direct effect of p53, wt and mutant p53 were prepared from E. coli-produced p53 and from insect cells infected with a recombinant baculovirus. When added into an in vitro transcription system, wt p53, but not mutant p53 reduced markedly transcription from the c-myc promoter, as well as from an array of other promoters, with the exception of an MHC class I gene promoter. The presence of wt p53 seemed to affect specifically the formation of the transcription preinitiation complex because preformed initiation complexes were completely refractory to wt p53, as was also the process of transcript elongation. Wild-type but not mutant p53 interfered with the stable binding of TBP and TFIIA to the TATA motif, although both wt and mutant p53 could associate in vitro with purified TBP. We propose that upon binding to TBP, wt but not mutant p53 specifically blocks the ability of TBP to engage in interactions required for efficient transcriptional initiation. This may account, at least in part, for the ability of excess wt p53 to inhibit cell proliferation and to interfere with neoplastic processes.