[Prognosis of affinity change of the TATA-binding protein to TATA-boxes upon polymorphisms of the human gene promoter TATA boxes].
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TATA box
TATA-binding protein
TATA-Box Binding Protein
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Among the main events of transcription initiation of TATA-containing genes in eukayotes are the recognition and binding of the TATA-box by the TATA-binding protein (TBP) to start the preinitiation complex formation on the nucleosomal DNA. Using the equilibrium equation for step-by-step TBP/TATA-binding, we have analyzed 69 experimental datasets on the characteristics of biologicacally important features altered by TATA-box mutations. Among these features, the TBP/TATA-complex parameters, the transcription level, the activity of gene products, yeast colony growth at a dose of growth inhibitor (phenotype), and the heterogenity of the response of a population to unspecific environmental stress have been described. Significant correlations were found between in silico prediction for TBP/TATA affinity and experimental data for in vivo and in vitro test-systems based on 15 cell types of 19 species, RNA polymerases II and III, and natural, recombinant or mutant TBP. Such an invariant impact of the step-by-step TBP/TATA-binding on the biological activity of complex systems, from a molecule to a population, might be due to the fact that TBP/TATA-complex formation precedes specific steps of transcription machinery assembly, which provide the multivariant jigsaw puzzle according to the expression pattern of each eukaryotic gene.
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Most RNA polymerase (Pol) II promoters lack a TATA element, yet nearly all Pol II transcription requires TATA binding protein (TBP). While the TBP-TATA interaction is critical for transcription at TATA-containing promoters, it has been unclear whether TBP sequence-specific DNA contacts are required for transcription at TATA-less genes. Transcription factor IID (TFIID), the TBP-containing coactivator that functions at most TATA-less genes, recognizes short sequence-specific promoter elements in metazoans, but analogous promoter elements have not been identified in Saccharomyces cerevisiae. We generated a set of mutations in the yeast TBP DNA binding surface and found that most support growth of yeast. Both in vivo and in vitro, many of these mutations are specifically defective for transcription of two TATA-containing genes with only minor defects in transcription of two TATA-less, TFIID-dependent genes. TBP binds several TATA-less promoters with apparent high affinity, but our results suggest that this binding is not important for transcription activity. Our results are consistent with the model that sequence-specific TBP-DNA contacts are not important at yeast TATA-less genes and suggest that other general transcription factors or coactivator subunits are responsible for recognition of TATA-less promoters. Our results also explain why yeast TBP derivatives defective for TATA binding appear defective in activated transcription.
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TATA box
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TATA-binding protein
CAAT box
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TATA box
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TATA-binding protein
RNA polymerase II
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General transcription factor
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The BTAF1 transcription factor interacts with TATA-binding protein (TBP) to form the B–TFIID complex, which is involved in RNA polymerase II transcription. Here, we present an extensive mapping study of TBP residues involved in BTAF1 interaction. This shows that residues in the concave, DNA-binding surface of TBP are important for BTAF1 binding. In addition, BTAF1 interacts with residues in helix 2 on the convex side of TBP as assayed in protein–protein and in DNA-binding assays. BTAF1 drastically changes the TATA-box binding specificity of TBP, as it is able to recruit DNA-binding defective TBP mutants to both TATA-containing and TATA-less DNA. Interestingly, other helix 2 interacting factors, such as TFIIA and NC2, can also stabilize mutant TBP binding to DNA. In contrast, TFIIB which interacts with a distinct surface of TBP does not display this activity. Since many proteins contact helix 2 of TBP, this provides a molecular basis for mutually exclusive TBP interactions and stresses the importance of this structural element for eukaryotic transcription.
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We have conducted a quantitative thermodynamic study of the effects of the TATA element and TATA-flanking sequences on the assembly of complexes containing TATA-binding protein (TBP) and the TFIIB-related factor, TFIIIB70. TBP binds to the sequence TATAAAAG in the context of the yeast U6 gene (yU6 hybrid TATA) or the adenovirus major late promoter (AdMLP) with different affinities demonstrating that the sequence context of a TATA element contributes to TBP binding. We also determined the cooperative free energies of formation of TBP.TFIIIB70.DNA complexes on the yU6 TATA element, the yU6 hybrid TATA element and a nonconsensus TATA element. The yU6 hybrid TATA displayed a moderate, less than 5-fold, increase in TBP affinity similar to the 3-fold increase observed for the AdMLP. In contrast, the nonconsensus and yU6 TATAs increased the affinity of TBP for DNA 12- and 17-fold, respectively. Since the TBP-TFIIIB70 cooperativity is greater on lower affinity TATA boxes and most polymerase III genes contain low affinity "TATA boxes," we conclude that the cooperative binding of TFIIIB70 and TBP to DNA represents an important driving force in the assembly of polymerase III-specific transcription complexes. An effect of the sequences surrounding the TATA box was also observed on TBP-TFIIIB70 cooperativity. The mechanistic implications of the thermodynamic linkage between DNA sequence and binding cooperativity are discussed.
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A yeast in vitro system was developed that is active for transcription at both TATA-containing and TATA-less promoters. Transcription with extracts made from cells depleted of TFIID subunit Taf1 demonstrated that promoters of both classes are TFIID dependent, in agreement with recent in vivo findings. TFIID depletion can be complemented in vitro by additional recombinant TATA binding protein (TBP) at only the TATA-containing promoters. In contrast, high levels of TBP did not complement Taf1 depletion in vivo and instead repressed transcription from both promoter types. We also demonstrate the importance of the TATA-like sequence found at many TATA-less promoters and describe how the presence or absence of the TATA element is likely not the only feature that distinguishes these two types of promoters.
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The human snRNA genes transcribed by RNA polymerase II (pol II) and III (pol III) have different core promoter elements. Both gene types contain similar proximal sequence elements (PSEs) but differ in the absence (pol II) or presence (pol III) of a TATA-box, which, together with the PSE, determines the assembly of a pol III-specific pre-initiation complex. BRFU is a factor exclusively required for transcription of the pol III-type snRNA genes. We report that recruitment of BRFU to the TATA-box of these promoters is TATA-binding protein (TBP)-dependent. BRFU in turn stabilizes TBP on TATA-containing template and extends the TBP footprint both upstream and downstream of the TATA element. The core domain of TBP is sufficient for BRFU·TBP·DNA complex formation and for interaction with BRFU off the template. We have mapped amino acid residues within TBP and domains of BRFU that mediate this interaction. BRFU has no specificity for sequences flanking the TATA-box and also forms a stable complex on the TATA-box of the pol II-specific adenovirus major late promoter (AdMLP). Furthermore, pol III-type transcription can initiate from an snRNA gene promoter containing an AdMLP TATA-box and flanking sequences. Therefore, the polymerase recruitment is not simply determined by the sequence of the TATA-box and immediate flanking sequences. The human snRNA genes transcribed by RNA polymerase II (pol II) and III (pol III) have different core promoter elements. Both gene types contain similar proximal sequence elements (PSEs) but differ in the absence (pol II) or presence (pol III) of a TATA-box, which, together with the PSE, determines the assembly of a pol III-specific pre-initiation complex. BRFU is a factor exclusively required for transcription of the pol III-type snRNA genes. We report that recruitment of BRFU to the TATA-box of these promoters is TATA-binding protein (TBP)-dependent. BRFU in turn stabilizes TBP on TATA-containing template and extends the TBP footprint both upstream and downstream of the TATA element. The core domain of TBP is sufficient for BRFU·TBP·DNA complex formation and for interaction with BRFU off the template. We have mapped amino acid residues within TBP and domains of BRFU that mediate this interaction. BRFU has no specificity for sequences flanking the TATA-box and also forms a stable complex on the TATA-box of the pol II-specific adenovirus major late promoter (AdMLP). Furthermore, pol III-type transcription can initiate from an snRNA gene promoter containing an AdMLP TATA-box and flanking sequences. Therefore, the polymerase recruitment is not simply determined by the sequence of the TATA-box and immediate flanking sequences. small nuclear RNA proximal sequence element TATA-binding protein pre-initiation complex glutathione S-transferase electrophoretic mobility shift assay adenovirus major late promoter polymerase II and III dithiothreitol bovine serum albumin polyacrylamide gel electrophoresis base pair(s) wild-type altered specificity C-terminal core domain of TBP The core promoter regions of human snRNA1 genes are sufficient to direct low levels of transcription in vitro and contain a binding site, called the proximal sequence element (PSE), for the multisubunit factor PBP/PTF/SNAPc (1Waldschmidt R. Wanandi I. Seifart K.H. EMBO J. 1991; 10: 2595-2603Crossref PubMed Scopus (101) Google Scholar, 2Murphy S. Yoon J.B. Gerster T. Roeder R.G. Mol. Cell. Biol. 1992; 12: 3247-3261Crossref PubMed Scopus (149) Google Scholar, 3Sadowski C.L. Henry R.W. Lobo S.M. Hernandez N. Genes Dev. 1993; 7: 1535-1548Crossref PubMed Scopus (141) Google Scholar). The PSE, usually located around −55, is interchangeable between snRNA gene promoters recognized by RNA polymerase II (e.g. U1 and U2) and RNA polymerase III (e.g. U6 and 7SK) (4Lobo, S. M., and Hernandez, N. T. (1994) inTranscription: Mechanisms and Regulation (Conaway, R. C., and Conaway, J. W., eds), pp. 127–159, Chapter 8, Raven Press, Ltd., New York.Google Scholar, 5Boyd D.C. Turner P.C. Watkins N.J. Gerster T. Murphy S. J. Mol. Biol. 1995; 253: 677-690Crossref PubMed Scopus (21) Google Scholar) and purified or recombinant PTF functions as a basal transcription factor for both types of snRNA gene (6Yoon J.B. Roeder R.G. Mol. Cell. Biol. 1996; 16: 1-9Crossref PubMed Google Scholar, 7Henry R.W. Mittal V. Ma B. Kobayashi R. Hernandez N. Genes Dev. 1998; 12: 2664-2672Crossref PubMed Scopus (63) Google Scholar). The pol III-specific core promoters contain an additional TATA-box at −25, which in this context is responsible for the selective recruitment of pol III (5Boyd D.C. Turner P.C. Watkins N.J. Gerster T. Murphy S. J. Mol. Biol. 1995; 253: 677-690Crossref PubMed Scopus (21) Google Scholar, 8Lobo S.M. Hernandez N. Cell. 1989; 58: 55-67Abstract Full Text PDF PubMed Scopus (174) Google Scholar). Insertion of a TATA element into the pol II-transcribed U2 promoter converts it into a predominantly pol III promoter (8Lobo S.M. Hernandez N. Cell. 1989; 58: 55-67Abstract Full Text PDF PubMed Scopus (174) Google Scholar), whereas mutation of the 7SK TATA-box reduces pol III transcription and allows snRNA-type transcription by pol II to occur (5Boyd D.C. Turner P.C. Watkins N.J. Gerster T. Murphy S. J. Mol. Biol. 1995; 253: 677-690Crossref PubMed Scopus (21) Google Scholar). TBP is required for transcription of both types of snRNA gene and is likely to be recruited to the TATA-less pol II-specific promoters by interaction with PTF binding to the PSE (3Sadowski C.L. Henry R.W. Lobo S.M. Hernandez N. Genes Dev. 1993; 7: 1535-1548Crossref PubMed Scopus (141) Google Scholar). PTF also potentiates direct binding of TBP to the TATA-box of the pol III-specific promoters (9Mittal V. Hernandez N. Science. 1997; 275: 1136-1140Crossref PubMed Scopus (76) Google Scholar). Because loss of pol III transcription correlates with the loss of TBP binding to the mutated TATA-box (5Boyd D.C. Turner P.C. Watkins N.J. Gerster T. Murphy S. J. Mol. Biol. 1995; 253: 677-690Crossref PubMed Scopus (21) Google Scholar, 10Lobo S.M. Lister J. Sullivan M.L. Hernandez N. Genes Dev. 1991; 5: 1477-1489Crossref PubMed Scopus (98) Google Scholar), the differential interaction of TBP with template DNA and the other proteins of the PIC is likely to play a key role in the ultimate recruitment of different polymerases.For transcription of tRNA and 5 S rRNA genes, which have gene-internal pol III promoters, TBP is associated with TFIIIB90 (11Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7026-7030Crossref PubMed Scopus (109) Google Scholar) (also called hBRF (12Mital R. Kobayashi R. Hernandez N. Mol. Cell. Biol. 1996; 16: 7031-7042Crossref PubMed Scopus (67) Google Scholar)) and hB“ (13Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (109) Google Scholar) (also called TFIIIB150 (14Teichmann M. Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14200-14205Crossref PubMed Scopus (67) Google Scholar)) within the TFIIIB-β complex (15Teichmann M. Seifart K.H. EMBO J. 1995; 14: 5974-5983Crossref PubMed Scopus (50) Google Scholar). At these TATA-less promoters, the internal promoter recruits TFIIIC that results in the subsequent recruitment of TFIIIB-β, which may then directly recruit pol III. TBP is a more loosely associated subunit of the less well characterized snRNA-specific TFIIIB form, designated hTFIIIB-α (15Teichmann M. Seifart K.H. EMBO J. 1995; 14: 5974-5983Crossref PubMed Scopus (50) Google Scholar), which is required for transcription of the U6/7SK genes by pol III. Recently, a basal transcription factor known as BRFU (13Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (109) Google Scholar), or TFIIIB50 (14Teichmann M. Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14200-14205Crossref PubMed Scopus (67) Google Scholar), has been shown to be required for transcription of these snRNA genes but not an adenovirus 2 VA1 gene with an internal pol III promoter. Interestingly, BRFU/TFIIIB50 has sequence homology to both TFIIIB90/BRF and the pol II initiation factor TFIIB. A complex of TFIIIB50 and four tightly associated factors constitutes, together with TBP and TFIIIB150, the complete TFIIIB-α activity that transcribes pol III snRNA genes (14Teichmann M. Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14200-14205Crossref PubMed Scopus (67) Google Scholar). However, Hernandez and colleagues (16Chong S.S. Hu P. Hernandez N. J. Biol. Chem. 2001; 276: 20727-20734Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) could obtain U6 transcription by combining a partially purified pol III fraction with recombinant PTF, TBP, hB”, and BRFU alone (16Chong S.S. Hu P. Hernandez N. J. Biol. Chem. 2001; 276: 20727-20734Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In addition, another factor encoded by an alternatively spliced variant of hBRF (BRF2) may also be required for U6 transcription (17McCulloch V. Hardin P. Peng W. Ruppert J.M. Lobo-Ruppert S.M. EMBO J. 2000; 19: 4134-4143Crossref PubMed Scopus (29) Google Scholar). Thus the exact “TFIIIB complex” requirement for pol III-transcribed snRNA genes remains to be determined.The ∼50-kDa human BRFU represents another member of the TFIIB-related protein family and has conserved zinc and core domains, and a divergent C-terminal domain (13Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (109) Google Scholar), (18Hernandez N. J. Biol. Chem. 2001; 276: 26733-26736Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Within the Zn2+-binding region, BRFU is 37.5 and 31.2% identical to human TFIIB and BRF, respectively, suggesting that this region of BRFU also adopts a zinc ribbon structure. The identity with the TFIIB core region is 19% (13Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (109) Google Scholar), and as in TFIIB, the BRFU core domain consists of two direct repeats.Here we show that BRFU interacts with TBP to form a complex on TATA-containing templates and have mapped amino acid residues within TBP and domains of BRFU that mediate this interaction. Strikingly, we found that BRFU, unlike TFIIB, appears to have no specificity for sequences outside the binding site for TBP. Together, the data presented here provide an insight into an important step in nucleation of a pol III- specific snRNA transcription initiation complex.DISCUSSIONThe exact mechanism of differential PIC assembly on pol II and pol III snRNA promoters is still awaiting elucidation. TBP is required for transcription of both types of snRNA genes but not as part of the TBP-containing complexes TFIID (32Bernues J. Simmen K.A. Lewis J.D. Gunderson S.I. Polycarpou-Schwarz M. Moncollin V. Egly J.M. Mattaj I.W. EMBO J. 1993; 12: 3573-3585Crossref PubMed Scopus (41) Google Scholar) or TFIIIB-β (3Sadowski C.L. Henry R.W. Lobo S.M. Hernandez N. Genes Dev. 1993; 7: 1535-1548Crossref PubMed Scopus (141) Google Scholar, 6Yoon J.B. Roeder R.G. Mol. Cell. Biol. 1996; 16: 1-9Crossref PubMed Google Scholar, 15Teichmann M. Seifart K.H. EMBO J. 1995; 14: 5974-5983Crossref PubMed Scopus (50) Google Scholar) that function in transcription of mRNA genes (by pol II) and tRNA/5 S RNA genes (by pol III), respectively. Because the same PTF is required for transcription of snRNA genes by both pol II and III, it seems likely that different modes of TBP recruitment by both PTF and promoter sequences set the stage for subsequent recruitment of polymerase-specific factors. Here we show that TBP bound to the TATA-box of pol III snRNA templates can recruit the pol III-specific snRNA-gene factor BRFU but not the pol II-specific TFIIB and that the intact TATA element is essential for BRFU·TBP·DNA complex formation. The stability of the BRFU·TBP·DNA complex resembles to some extent the yeast TFIIIB complex, which consists of strongly associated TBP and BRF and loosely associated B“ (33Kassavetis G.A. Bartholomew B. Blanco J.A. Johnson T.E. Geiduschek E.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7308-7312Crossref PubMed Scopus (102) Google Scholar, 34Huet J. Conesa C. Manaud N. Chaussivert N. Sentenac A. Nucleic Acids Res. 1994; 22: 3433-3439Crossref PubMed Scopus (30) Google Scholar). The non-conserved N-terminal domain of TBP was reported to be responsible for its cooperative binding with PTF to their respective binding sites on U6 promoter (9Mittal V. Hernandez N. Science. 1997; 275: 1136-1140Crossref PubMed Scopus (76) Google Scholar). In contrast, we find that the C-terminal core domain of TBP (cTBP) is sufficient to mediate interaction with BRFU both in solution and on DNA.We could not detect direct interaction between BRFU and DNA, but DNase I analysis suggests that BRFU contacts sequences upstream and downstream of the TATA-box when TBP is bound. These sequences might play a role in the clamping of TBP to DNA by BRFU. We speculate there is a TBP-induced DNA bend in BRFU·TBP·DNA complex that places the upstream and downstream DNA segments in proper spatial register for simultaneous BRFU·DNA interactions with both DNA segments. It is therefore possible that, like TFIIB (27Tsai F.T. Sigler P.B. EMBO J. 2000; 19: 25-36Crossref PubMed Scopus (139) Google Scholar), binding of BRFU is synergetic with TBP requiring the distortion of the TATA-box (Fig. 2 B). Biochemical studies have shown that cTBP recognizes the TATA-box of protein-encoding genes in both orientations (reviewed in Ref. 35Reinberg D. Orphanides G. Ebright R. Akoulitchev S. Carcamo J. Cho H. Cortes P. Drapkin R. Flores O. Ha I. Inostroza J.A. Kim S. Kim T.K. Kumar P. Lagrange T. LeRoy G. Lu H. Ma D.M. Maldonado E. Merino A. Mermelstein F. Olave I. Sheldon M. Shiekhattar R. Zawel L. et al.Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 83-103Crossref PubMed Scopus (55) Google Scholar) and TFIIB, as an essential factor in the assembly of a functional PIC, forms a stereo-specific complex with TBP (36Tansey W.P. Herr W. Science. 1997; 275: 829-831Crossref PubMed Scopus (38) Google Scholar). It, therefore, follows that a specific TFIIB·BRE interaction (31Lagrange T. Kapanidis A.N. Tang H. Reinberg D. Ebright R.H. Genes Dev. 1998; 12: 34-44Crossref PubMed Scopus (304) Google Scholar) would contribute strongly to unique directionality in the assembly of the PIC and, hence, to the polarity of transcription. In the yeast Saccharomyces cerevisiae, TFIIIB is recruited to the U6 promoter through the interaction of its TBP subunit with a TATA-box, and the direction of this complex assembly is dictated by a TFIIIC-dependent mechanism (37Whitehall S.K. Kassavetis G.A. Geiduschek E.P. Genes Dev. 1995; 9: 2974-2985Crossref PubMed Scopus (59) Google Scholar). It remains to be determined whether the sequences flanking the TATA-box in human pol III-specific snRNA templates play a role in setting the orientation of the TBP·BRFU complex.Although the BRFU core possesses a structure similar to the core of TFIIB, we can expect differences in composition of TFIIB·TBP·DNA and BRFU·TBP·DNA complexes. The TFIIB C-terminal domain, containing intact direct repeats and associated basic regions, is necessary for interaction with TBP·DNA complexes (30Hisatake K. Roeder R.G. Horikoshi M. Nature. 1993; 363: 744-747Crossref PubMed Scopus (58) Google Scholar). We found that direct repeat 2 of the BRFU core, but not repeat 1, is required for formation of a TBP·BRFU·DNA complex. In hBRF, both repeats and more avidly the C-terminal half of the protein interact with TBP (11Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7026-7030Crossref PubMed Scopus (109) Google Scholar). The transcriptionally active BRF2 variant is also able to form a complex with TBP (17McCulloch V. Hardin P. Peng W. Ruppert J.M. Lobo-Ruppert S.M. EMBO J. 2000; 19: 4134-4143Crossref PubMed Scopus (29) Google Scholar). In this regard it should be noted that the 23-kDa BRF2 does not contain all of the structural regions that are typical for TFIIB-related proteins (zinc-ribbon, repeat 1, and repeat 2) and includes only part of the second direct repeat. In TFIIB, the zinc-ribbon domain is required for direct recruitment of a TFIIF·pol II complex (38Ha I. Roberts S. Maldonado E. Sun X. Kim L.U. Green M. Reinberg D. Genes Dev. 1993; 7: 1021-1032Crossref PubMed Scopus (167) Google Scholar, 39Fang S.M. Burton Z.F. J. Biol. Chem. 1996; 271: 11703-11709Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). BRF, like TFIIB, also directly contacts polymerase, in this case pol III (11Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7026-7030Crossref PubMed Scopus (109) Google Scholar, 40Khoo B. Brophy B. Jackson S.P. Genes Dev. 1994; 8: 2879-2890Crossref PubMed Scopus (108) Google Scholar). However, the Zn-ribbon in BRF plays a role in open complex formation in yeast (41Hahn S. Roberts S. Genes Dev. 2000; 14: 719-730PubMed Google Scholar) but is not required for pol III recruitment (41Hahn S. Roberts S. Genes Dev. 2000; 14: 719-730PubMed Google Scholar, 42Kassavetis G.A. Bardeleben C. Kumar A. Ramirez E. Geiduschek E.P. Mol. Cell. Biol. 1997; 17: 5299-5306Crossref PubMed Scopus (45) Google Scholar, 43Kassavetis G.A. Kumar A. Letts G.A. Geiduschek E.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9196-9201Crossref PubMed Scopus (53) Google Scholar). Further experiments are therefore required to reveal the precise function of the Zn-ribbon in BRFU.Earlier studies of TBP mutants have already revealed a great deal about how the protein functions in pol II and pol III transcription. Residues Glu-284, Glu-286, and Leu-287 at the tip of the second stirrup of the saddle-shaped molecule (44Kim J.L. Burley S.K. Nat. Struct. Biol. 1994; 1: 638-653Crossref PubMed Scopus (198) Google Scholar) are critical both for TFIIB binding and in vitro and in vivo transcription (19Bryant G.O. Martel L.S. Burley S.K. Berk A.J. Genes Dev. 1996; 10: 2491-2504Crossref PubMed Scopus (99) Google Scholar, 28Tang H. Sun X. Reinberg D. Ebright R.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1119-1124Crossref PubMed Scopus (93) Google Scholar,45Kim T.K. Hashimoto S. Kelleher 3rd, R.J. Flanagan P.M. Kornberg R.D. Horikoshi M. Roeder R.G. Nature. 1994; 369: 252-255Crossref PubMed Scopus (101) Google Scholar). In yeast, different residues Arg-231, Arg-235, and Phe-250 contribute to the TBP surface that interacts with BRF (29Shen Y. Kassavetis G.A. Bryant G.O. Berk A.J. Mol. Cell. Biol. 1998; 18: 1692-1700Crossref PubMed Scopus (43) Google Scholar). In our assays, single-amino acid substitutions R235E and F250E in TBP prevent BRFU from entering a TBP·DNA complex. Mutation E284R did not affect the stability of the BRFU·TBP·DNA complex but caused significant changes in the complex conformation. Thus, these data are consistent with the BRFU sequence similarities to both BRF and TFIIB.Clearly, the sequences flanking the TATA-box in the U2 TATA construct do not allow TBP·TFIIB·DNA complex formation and thus might exclude TFIIF-pol II recruitment, and in consequence, pol II-specific transcription. However, because BRFU forms a complex with TBP·AdMLP as well as TFIIB does and pol III-type transcription can initiate from an snRNA gene promoter containing an mRNA gene TATA-box and flanking sequences, additional mechanism(s) must direct BRFU to specifically assemble on pol III snRNA templates in the cell. Otherwise, BRFU would compete with TFIIB in PIC formation on the promoters of protein-encoding genes (Fig.7). Thus, sequences further outside the core promoter of the AdMLP may ensure that only pol II-specific PICs can form on this template in vivo. For instance, factors binding to promoter and initiator sequences may interact specifically with pol II-specific basic factors and effectively exclude pol III-specific factors. However, at least one mRNA promoter, within the c-myc gene, can direct TATA-dependent transcription by pol III both in vitro and in vivo in some circumstances (46Sussman D.J. Chung J. Leder P. Nucleic Acids Res. 1991; 19: 5045-5052Crossref PubMed Scopus (18) Google Scholar), suggesting that the balance can be tipped to favor pol III.The availability of individual factors, TBP, PTF, B“, and pol III snRNA-specific BRFU offers a unique system to understand the structure and function of a basal transcription multisubunit complex specific for snRNA genes transcribed by pol III polymerase. It is possible that, in addition to TBP, BRFU or its associated factors directly contact transcription factors PTF and B” and, together with any recognition elements in the core of pol III snRNA promoters, provides a basis for selective recruitment of pol III. The core promoter regions of human snRNA1 genes are sufficient to direct low levels of transcription in vitro and contain a binding site, called the proximal sequence element (PSE), for the multisubunit factor PBP/PTF/SNAPc (1Waldschmidt R. Wanandi I. Seifart K.H. EMBO J. 1991; 10: 2595-2603Crossref PubMed Scopus (101) Google Scholar, 2Murphy S. Yoon J.B. Gerster T. Roeder R.G. Mol. Cell. Biol. 1992; 12: 3247-3261Crossref PubMed Scopus (149) Google Scholar, 3Sadowski C.L. Henry R.W. Lobo S.M. Hernandez N. Genes Dev. 1993; 7: 1535-1548Crossref PubMed Scopus (141) Google Scholar). The PSE, usually located around −55, is interchangeable between snRNA gene promoters recognized by RNA polymerase II (e.g. U1 and U2) and RNA polymerase III (e.g. U6 and 7SK) (4Lobo, S. M., and Hernandez, N. T. (1994) inTranscription: Mechanisms and Regulation (Conaway, R. C., and Conaway, J. W., eds), pp. 127–159, Chapter 8, Raven Press, Ltd., New York.Google Scholar, 5Boyd D.C. Turner P.C. Watkins N.J. Gerster T. Murphy S. J. Mol. Biol. 1995; 253: 677-690Crossref PubMed Scopus (21) Google Scholar) and purified or recombinant PTF functions as a basal transcription factor for both types of snRNA gene (6Yoon J.B. Roeder R.G. Mol. Cell. Biol. 1996; 16: 1-9Crossref PubMed Google Scholar, 7Henry R.W. Mittal V. Ma B. Kobayashi R. Hernandez N. Genes Dev. 1998; 12: 2664-2672Crossref PubMed Scopus (63) Google Scholar). The pol III-specific core promoters contain an additional TATA-box at −25, which in this context is responsible for the selective recruitment of pol III (5Boyd D.C. Turner P.C. Watkins N.J. Gerster T. Murphy S. J. Mol. Biol. 1995; 253: 677-690Crossref PubMed Scopus (21) Google Scholar, 8Lobo S.M. Hernandez N. Cell. 1989; 58: 55-67Abstract Full Text PDF PubMed Scopus (174) Google Scholar). Insertion of a TATA element into the pol II-transcribed U2 promoter converts it into a predominantly pol III promoter (8Lobo S.M. Hernandez N. Cell. 1989; 58: 55-67Abstract Full Text PDF PubMed Scopus (174) Google Scholar), whereas mutation of the 7SK TATA-box reduces pol III transcription and allows snRNA-type transcription by pol II to occur (5Boyd D.C. Turner P.C. Watkins N.J. Gerster T. Murphy S. J. Mol. Biol. 1995; 253: 677-690Crossref PubMed Scopus (21) Google Scholar). TBP is required for transcription of both types of snRNA gene and is likely to be recruited to the TATA-less pol II-specific promoters by interaction with PTF binding to the PSE (3Sadowski C.L. Henry R.W. Lobo S.M. Hernandez N. Genes Dev. 1993; 7: 1535-1548Crossref PubMed Scopus (141) Google Scholar). PTF also potentiates direct binding of TBP to the TATA-box of the pol III-specific promoters (9Mittal V. Hernandez N. Science. 1997; 275: 1136-1140Crossref PubMed Scopus (76) Google Scholar). Because loss of pol III transcription correlates with the loss of TBP binding to the mutated TATA-box (5Boyd D.C. Turner P.C. Watkins N.J. Gerster T. Murphy S. J. Mol. Biol. 1995; 253: 677-690Crossref PubMed Scopus (21) Google Scholar, 10Lobo S.M. Lister J. Sullivan M.L. Hernandez N. Genes Dev. 1991; 5: 1477-1489Crossref PubMed Scopus (98) Google Scholar), the differential interaction of TBP with template DNA and the other proteins of the PIC is likely to play a key role in the ultimate recruitment of different polymerases. For transcription of tRNA and 5 S rRNA genes, which have gene-internal pol III promoters, TBP is associated with TFIIIB90 (11Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7026-7030Crossref PubMed Scopus (109) Google Scholar) (also called hBRF (12Mital R. Kobayashi R. Hernandez N. Mol. Cell. Biol. 1996; 16: 7031-7042Crossref PubMed Scopus (67) Google Scholar)) and hB“ (13Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (109) Google Scholar) (also called TFIIIB150 (14Teichmann M. Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14200-14205Crossref PubMed Scopus (67) Google Scholar)) within the TFIIIB-β complex (15Teichmann M. Seifart K.H. EMBO J. 1995; 14: 5974-5983Crossref PubMed Scopus (50) Google Scholar). At these TATA-less promoters, the internal promoter recruits TFIIIC that results in the subsequent recruitment of TFIIIB-β, which may then directly recruit pol III. TBP is a more loosely associated subunit of the less well characterized snRNA-specific TFIIIB form, designated hTFIIIB-α (15Teichmann M. Seifart K.H. EMBO J. 1995; 14: 5974-5983Crossref PubMed Scopus (50) Google Scholar), which is required for transcription of the U6/7SK genes by pol III. Recently, a basal transcription factor known as BRFU (13Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (109) Google Scholar), or TFIIIB50 (14Teichmann M. Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14200-14205Crossref PubMed Scopus (67) Google Scholar), has been shown to be required for transcription of these snRNA genes but not an adenovirus 2 VA1 gene with an internal pol III promoter. Interestingly, BRFU/TFIIIB50 has sequence homology to both TFIIIB90/BRF and the pol II initiation factor TFIIB. A complex of TFIIIB50 and four tightly associated factors constitutes, together with TBP and TFIIIB150, the complete TFIIIB-α activity that transcribes pol III snRNA genes (14Teichmann M. Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14200-14205Crossref PubMed Scopus (67) Google Scholar). However, Hernandez and colleagues (16Chong S.S. Hu P. Hernandez N. J. Biol. Chem. 2001; 276: 20727-20734Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) could obtain U6 transcription by combining a partially purified pol III fraction with recombinant PTF, TBP, hB”, and BRFU alone (16Chong S.S. Hu P. Hernandez N. J. Biol. Chem. 2001; 276: 20727-20734Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In addition, another factor encoded by an alternatively spliced variant of hBRF (BRF2) may also be required for U6 transcription (17McCulloch V. Hardin P. Peng W. Ruppert J.M. Lobo-Ruppert S.M. EMBO J. 2000; 19: 4134-4143Crossref PubMed Scopus (29) Google Scholar). Thus the exact “TFIIIB complex” requirement for pol III-transcribed snRNA genes remains to be determined. The ∼50-kDa human BRFU represents another member of the TFIIB-related protein family and has conserved zinc and core domains, and a divergent C-terminal domain (13Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (109) Google Scholar), (18Hernandez N. J. Biol. Chem. 2001; 276: 26733-26736Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Within the Zn2+-binding region, BRFU is 37.5 and 31.2% identical to human TFIIB and BRF, respectively, suggesting that this region of BRFU also adopts a zinc ribbon structure. The identity with the TFIIB core region is 19% (13Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (109) Google Scholar), and as in TFIIB, the BRFU core domain consists of two direct repeats. Here we show that BRFU interacts with TBP to form a complex on TATA-containing templates and have mapped amino acid residues within TBP and domains of BRFU that mediate this interaction. Strikingly, we found that BRFU, unlike TFIIB, appears to have no specificity for sequences outside the binding site for TBP. Together, the data presented here provide an insight into an important step in nucleation of a pol III- specific snRNA transcription initiation complex. DISCUSSIONThe exact mechanism of differential PIC assembly on pol II and pol III snRNA promoters is still awaiting elucidation. TBP is required for transcription of both types of snRNA genes but not as part of the TBP-containing complexes TFIID (32Bernues J. Simmen K.A. Lewis J.D. Gunderson S.I. Polycarpou-Schwarz M. Moncollin V. Egly J.M. Mattaj I.W. EMBO J. 1993; 12: 3573-3585Crossref PubMed Scopus (41) Google Scholar) or TFIIIB-β (3Sadowski C.L. Henry R.W. Lobo S.M. Hernandez N. Genes Dev. 1993; 7: 1535-1548Crossref PubMed Scopus (141) Google Scholar, 6Yoon J.B. Roeder R.G. Mol. Cell. Biol. 1996; 16: 1-9Crossref PubMed Google Scholar, 15Teichmann M. Seifart K.H. EMBO J. 1995; 14: 5974-5983Crossref PubMed Scopus (50) Google Scholar) that function in transcription of mRNA genes (by pol II) and tRNA/5 S RNA genes (by pol III), respectively. Because the same PTF is required for transcription of snRNA genes by both pol II and III, it seems likely that different modes of TBP recruitment by both PTF and promoter sequences set the stage for subsequent recruitment of polymerase-specific factors. Here we show that TBP bound to the TATA-box of pol III snRNA templates can recruit the pol III-specific snRNA-gene factor BRFU but not the pol II-specific TFIIB and that the intact TATA element is essential for BRFU·TBP·DNA complex formation. The stability of the BRFU·TBP·DNA complex resembles to some extent the yeast TFIIIB complex, which consists of strongly associated TBP and BRF and loosely associated B“ (33Kassavetis G.A. Bartholomew B. Blanco J.A. Johnson T.E. Geiduschek E.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7308-7312Crossref PubMed Scopus (102) Google Scholar, 34Huet J. Conesa C. Manaud N. Chaussivert N. Sentenac A. Nucleic Acids Res. 1994; 22: 3433-3439Crossref PubMed Scopus (30) Google Scholar). The non-conserved N-terminal domain of TBP was reported to be responsible for its cooperative binding with PTF to their respective binding sites on U6 promoter (9Mittal V. Hernandez N. Science. 1997; 275: 1136-1140Crossref PubMed Scopus (76) Google Scholar). In contrast, we find that the C-terminal core domain of TBP (cTBP) is sufficient to mediate interaction with BRFU both in solution and on DNA.We could not detect direct interaction between BRFU and DNA, but DNase I analysis suggests that BRFU contacts sequences upstream and downstream of the TATA-box when TBP is bound. These sequences might play a role in the clamping of TBP to DNA by BRFU. We speculate there is a TBP-induced DNA bend in BRFU·TBP·DNA complex that places the upstream and downstream DNA segments in proper spatial register for simultaneous BRFU·DNA interactions with both DNA segments. It is therefore possible that, like TFIIB (27Tsai F.T. Sigler P.B. EMBO J. 2000; 19: 25-36Crossref PubMed Scopus (139) Google Scholar), binding of BRFU is synergetic with TBP requiring the distortion of the TATA-box (Fig. 2 B). Biochemical studies have shown that cTBP recognizes the TATA-box of protein-encoding genes in both orientations (reviewed in Ref. 35Reinberg D. Orphanides G. Ebright R. Akoulitchev S. Carcamo J. Cho H. Cortes P. Drapkin R. Flores O. Ha I. Inostroza J.A. Kim S. Kim T.K. Kumar P. Lagrange T. LeRoy G. Lu H. Ma D.M. Maldonado E. Merino A. Mermelstein F. Olave I. Sheldon M. Shiekhattar R. Zawel L. et al.Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 83-103Crossref PubMed Scopus (55) Google Scholar) and TFIIB, as an essential factor in the assembly of a functional PIC, forms a stereo-specific complex with TBP (36Tansey W.P. Herr W. Science. 1997; 275: 829-831Crossref PubMed Scopus (38) Google Scholar). It, therefore, follows that a specific TFIIB·BRE interaction (31Lagrange T. Kapanidis A.N. Tang H. Reinberg D. Ebright R.H. Genes Dev. 1998; 12: 34-44Crossref PubMed Scopus (304) Google Scholar) would contribute strongly to unique directionality in the assembly of the PIC and, hence, to the polarity of transcription. In the yeast Saccharomyces cerevisiae, TFIIIB is recruited to the U6 promoter through the interaction of its TBP subunit with a TATA-box, and the direction of this complex assembly is dictated by a TFIIIC-dependent mechanism (37Whitehall S.K. Kassavetis G.A. Geiduschek E.P. Genes Dev. 1995; 9: 2974-2985Crossref PubMed Scopus (59) Google Scholar). It remains to be determined whether the sequences flanking the TATA-box in human pol III-specific snRNA templates play a role in setting the orientation of the TBP·BRFU complex.Although the BRFU core possesses a structure similar to the core of TFIIB, we can expect differences in composition of TFIIB·TBP·DNA and BRFU·TBP·DNA complexes. The TFIIB C-terminal domain, containing intact direct repeats and associated basic regions, is necessary for interaction with TBP·DNA complexes (30Hisatake K. Roeder R.G. Horikoshi M. Nature. 1993; 363: 744-747Crossref PubMed Scopus (58) Google Scholar). We found that direct repeat 2 of the BRFU core, but not repeat 1, is required for formation of a TBP·BRFU·DNA complex. In hBRF, both repeats and more avidly the C-terminal half of the protein interact with TBP (11Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7026-7030Crossref PubMed Scopus (109) Google Scholar). The transcriptionally active BRF2 variant is also able to form a complex with TBP (17McCulloch V. Hardin P. Peng W. Ruppert J.M. Lobo-Ruppert S.M. EMBO J. 2000; 19: 4134-4143Crossref PubMed Scopus (29) Google Scholar). In this regard it should be noted that the 23-kDa BRF2 does not contain all of the structural regions that are typical for TFIIB-related proteins (zinc-ribbon, repeat 1, and repeat 2) and includes only part of the second direct repeat. In TFIIB, the zinc-ribbon domain is required for direct recruitment of a TFIIF·pol II complex (38Ha I. Roberts S. Maldonado E. Sun X. Kim L.U. Green M. Reinberg D. Genes Dev. 1993; 7: 1021-1032Crossref PubMed Scopus (167) Google Scholar, 39Fang S.M. Burton Z.F. J. Biol. Chem. 1996; 271: 11703-11709Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). BRF, like TFIIB, also directly contacts polymerase, in this case pol III (11Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7026-7030Crossref PubMed Scopus (109) Google Scholar, 40Khoo B. Brophy B. Jackson S.P. Genes Dev. 1994; 8: 2879-2890Crossref PubMed Scopus (108) Google Scholar). However, the Zn-ribbon in BRF plays a role in open complex formation in yeast (41Hahn S. Roberts S. Genes Dev. 2000; 14: 719-730PubMed Google Scholar) but is not required for pol III recruitment (41Hahn S. Roberts S. Genes Dev. 2000; 14: 719-730PubMed Google Scholar, 42Kassavetis G.A. Bardeleben C. Kumar A. Ramirez E. Geiduschek E.P. Mol. Cell. Biol. 1997; 17: 5299-5306Crossref PubMed Scopus (45) Google Scholar, 43Kassavetis G.A. Kumar A. Letts G.A. Geiduschek E.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9196-9201Crossref PubMed Scopus (53) Google Scholar). Further experiments are therefore required to reveal the precise function of the Zn-ribbon in BRFU.Earlier studies of TBP mutants have already revealed a great deal about how the protein functions in pol II and pol III transcription. Residues Glu-284, Glu-286, and Leu-287 at the tip of the second stirrup of the saddle-shaped molecule (44Kim J.L. Burley S.K. Nat. Struct. Biol. 1994; 1: 638-653Crossref PubMed Scopus (198) Google Scholar) are critical both for TFIIB binding and in vitro and in vivo transcription (19Bryant G.O. Martel L.S. Burley S.K. Berk A.J. Genes Dev. 1996; 10: 2491-2504Crossref PubMed Scopus (99) Google Scholar, 28Tang H. Sun X. Reinberg D. Ebright R.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1119-1124Crossref PubMed Scopus (93) Google Scholar,45Kim T.K. Hashimoto S. Kelleher 3rd, R.J. Flanagan P.M. Kornberg R.D. Horikoshi M. Roeder R.G. Nature. 1994; 369: 252-255Crossref PubMed Scopus (101) Google Scholar). In yeast, different residues Arg-231, Arg-235, and Phe-250 contribute to the TBP surface that interacts with BRF (29Shen Y. Kassavetis G.A. Bryant G.O. Berk A.J. Mol. Cell. Biol. 1998; 18: 1692-1700Crossref PubMed Scopus (43) Google Scholar). In our assays, single-amino acid substitutions R235E and F250E in TBP prevent BRFU from entering a TBP·DNA complex. Mutation E284R did not affect the stability of the BRFU·TBP·DNA complex but caused significant changes in the complex conformation. Thus, these data are consistent with the BRFU sequence similarities to both BRF and TFIIB.Clearly, the sequences flanking the TATA-box in the U2 TATA construct do not allow TBP·TFIIB·DNA complex formation and thus might exclude TFIIF-pol II recruitment, and in consequence, pol II-specific transcription. However, because BRFU forms a complex with TBP·AdMLP as well as TFIIB does and pol III-type transcription can initiate from an snRNA gene promoter containing an mRNA gene TATA-box and flanking sequences, additional mechanism(s) must direct BRFU to specifically assemble on pol III snRNA templates in the cell. Otherwise, BRFU would compete with TFIIB in PIC formation on the promoters of protein-encoding genes (Fig.7). Thus, sequences further outside the core promoter of the AdMLP may ensure that only pol II-specific PICs can form on this template in vivo. For instance, factors binding to promoter and initiator sequences may interact specifically with pol II-specific basic factors and effectively exclude pol III-specific factors. However, at least one mRNA promoter, within the c-myc gene, can direct TATA-dependent transcription by pol III both in vitro and in vivo in some circumstances (46Sussman D.J. Chung J. Leder P. Nucleic Acids Res. 1991; 19: 5045-5052Crossref PubMed Scopus (18) Google Scholar), suggesting that the balance can be tipped to favor pol III.The availability of individual factors, TBP, PTF, B“, and pol III snRNA-specific BRFU offers a unique system to understand the structure and function of a basal transcription multisubunit complex specific for snRNA genes transcribed by pol III polymerase. It is possible that, in addition to TBP, BRFU or its associated factors directly contact transcription factors PTF and B” and, together with any recognition elements in the core of pol III snRNA promoters, provides a basis for selective recruitment of pol III. The exact mechanism of differential PIC assembly on pol II and pol III snRNA promoters is still awaiting elucidation. TBP is required for transcription of both types of snRNA genes but not as part of the TBP-containing complexes TFIID (32Bernues J. Simmen K.A. Lewis J.D. Gunderson S.I. Polycarpou-Schwarz M. Moncollin V. Egly J.M. Mattaj I.W. EMBO J. 1993; 12: 3573-3585Crossref PubMed Scopus (41) Google Scholar) or TFIIIB-β (3Sadowski C.L. Henry R.W. Lobo S.M. Hernandez N. Genes Dev. 1993; 7: 1535-1548Crossref PubMed Scopus (141) Google Scholar, 6Yoon J.B. Roeder R.G. Mol. Cell. Biol. 1996; 16: 1-9Crossref PubMed Google Scholar, 15Teichmann M. Seifart K.H. EMBO J. 1995; 14: 5974-5983Crossref PubMed Scopus (50) Google Scholar) that function in transcription of mRNA genes (by pol II) and tRNA/5 S RNA genes (by pol III), respectively. Because the same PTF is required for transcription of snRNA genes by both pol II and III, it seems likely that different modes of TBP recruitment by both PTF and promoter sequences set the stage for subsequent recruitment of polymerase-specific factors. Here we show that TBP bound to the TATA-box of pol III snRNA templates can recruit the pol III-specific snRNA-gene factor BRFU but not the pol II-specific TFIIB and that the intact TATA element is essential for BRFU·TBP·DNA complex formation. The stability of the BRFU·TBP·DNA complex resembles to some extent the yeast TFIIIB complex, which consists of strongly associated TBP and BRF and loosely associated B“ (33Kassavetis G.A. Bartholomew B. Blanco J.A. Johnson T.E. Geiduschek E.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7308-7312Crossref PubMed Scopus (102) Google Scholar, 34Huet J. Conesa C. Manaud N. Chaussivert N. Sentenac A. Nucleic Acids Res. 1994; 22: 3433-3439Crossref PubMed Scopus (30) Google Scholar). The non-conserved N-terminal domain of TBP was reported to be responsible for its cooperative binding with PTF to their respective binding sites on U6 promoter (9Mittal V. Hernandez N. Science. 1997; 275: 1136-1140Crossref PubMed Scopus (76) Google Scholar). In contrast, we find that the C-terminal core domain of TBP (cTBP) is sufficient to mediate interaction with BRFU both in solution and on DNA. We could not detect direct interaction between BRFU and DNA, but DNase I analysis suggests that BRFU contacts sequences upstream and downstream of the TATA-box when TBP is bound. These sequences might play a role in the clamping of TBP to DNA by BRFU. We speculate there is a TBP-induced DNA bend in BRFU·TBP·DNA complex that places the upstream and downstream DNA segments in proper spatial register for simultaneous BRFU·DNA interactions with both DNA segments. It is therefore possible that, like TFIIB (27Tsai F.T. Sigler P.B. EMBO J. 2000; 19: 25-36Crossref PubMed Scopus (139) Google Scholar), binding of BRFU is synergetic with TBP requiring the distortion of the TATA-box (Fig. 2 B). Biochemical studies have shown that cTBP recognizes the TATA-box of protein-encoding genes in both orientations (reviewed in Ref. 35Reinberg D. Orphanides G. Ebright R. Akoulitchev S. Carcamo J. Cho H. Cortes P. Drapkin R. Flores O. Ha I. Inostroza J.A. Kim S. Kim T.K. Kumar P. Lagrange T. LeRoy G. Lu H. Ma D.M. Maldonado E. Merino A. Mermelstein F. Olave I. Sheldon M. Shiekhattar R. Zawel L. et al.Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 83-103Crossref PubMed Scopus (55) Google Scholar) and TFIIB, as an essential factor in the assembly of a functional PIC, forms a stereo-specific complex with TBP (36Tansey W.P. Herr W. Science. 1997; 275: 829-831Crossref PubMed Scopus (38) Google Scholar). It, therefore, follows that a specific TFIIB·BRE interaction (31Lagrange T. Kapanidis A.N. Tang H. Reinberg D. Ebright R.H. Genes Dev. 1998; 12: 34-44Crossref PubMed Scopus (304) Google Scholar) would contribute strongly to unique directionality in the assembly of the PIC and, hence, to the polarity of transcription. In the yeast Saccharomyces cerevisiae, TFIIIB is recruited to the U6 promoter through the interaction of its TBP subunit with a TATA-box, and the direction of this complex assembly is dictated by a TFIIIC-dependent mechanism (37Whitehall S.K. Kassavetis G.A. Geiduschek E.P. Genes Dev. 1995; 9: 2974-2985Crossref PubMed Scopus (59) Google Scholar). It remains to be determined whether the sequences flanking the TATA-box in human pol III-specific snRNA templates play a role in setting the orientation of the TBP·BRFU complex. Although the BRFU core possesses a structure similar to the core of TFIIB, we can expect differences in composition of TFIIB·TBP·DNA and BRFU·TBP·DNA complexes. The TFIIB C-terminal domain, containing intact direct repeats and associated basic regions, is necessary for interaction with TBP·DNA complexes (30Hisatake K. Roeder R.G. Horikoshi M. Nature. 1993; 363: 744-747Crossref PubMed Scopus (58) Google Scholar). We found that direct repeat 2 of the BRFU core, but not repeat 1, is required for formation of a TBP·BRFU·DNA complex. In hBRF, both repeats and more avidly the C-terminal half of the protein interact with TBP (11Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7026-7030Crossref PubMed Scopus (109) Google Scholar). The transcriptionally active BRF2 variant is also able to form a complex with TBP (17McCulloch V. Hardin P. Peng W. Ruppert J.M. Lobo-Ruppert S.M. EMBO J. 2000; 19: 4134-4143Crossref PubMed Scopus (29) Google Scholar). In this regard it should be noted that the 23-kDa BRF2 does not contain all of the structural regions that are typical for TFIIB-related proteins (zinc-ribbon, repeat 1, and repeat 2) and includes only part of the second direct repeat. In TFIIB, the zinc-ribbon domain is required for direct recruitment of a TFIIF·pol II complex (38Ha I. Roberts S. Maldonado E. Sun X. Kim L.U. Green M. Reinberg D. Genes Dev. 1993; 7: 1021-1032Crossref PubMed Scopus (167) Google Scholar, 39Fang S.M. Burton Z.F. J. Biol. Chem. 1996; 271: 11703-11709Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). BRF, like TFIIB, also directly contacts polymerase, in this case pol III (11Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7026-7030Crossref PubMed Scopus (109) Google Scholar, 40Khoo B. Brophy B. Jackson S.P. Genes Dev. 1994; 8: 2879-2890Crossref PubMed Scopus (108) Google Scholar). However, the Zn-ribbon in BRF plays a role in open complex formation in yeast (41Hahn S. Roberts S. Genes Dev. 2000; 14: 719-730PubMed Google Scholar) but is not required for pol III recruitment (41Hahn S. Roberts S. Genes Dev. 2000; 14: 719-730PubMed Google Scholar, 42Kassavetis G.A. Bardeleben C. Kumar A. Ramirez E. Geiduschek E.P. Mol. Cell. Biol. 1997; 17: 5299-5306Crossref PubMed Scopus (45) Google Scholar, 43Kassavetis G.A. Kumar A. Letts G.A. Geiduschek E.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9196-9201Crossref PubMed Scopus (53) Google Scholar). Further experiments are therefore required to reveal the precise function of the Zn-ribbon in BRFU. Earlier studies of TBP mutants have already revealed a great deal about how the protein functions in pol II and pol III transcription. Residues Glu-284, Glu-286, and Leu-287 at the tip of the second stirrup of the saddle-shaped molecule (44Kim J.L. Burley S.K. Nat. Struct. Biol. 1994; 1: 638-653Crossref PubMed Scopus (198) Google Scholar) are critical both for TFIIB binding and in vitro and in vivo transcription (19Bryant G.O. Martel L.S. Burley S.K. Berk A.J. Genes Dev. 1996; 10: 2491-2504Crossref PubMed Scopus (99) Google Scholar, 28Tang H. Sun X. Reinberg D. Ebright R.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1119-1124Crossref PubMed Scopus (93) Google Scholar,45Kim T.K. Hashimoto S. Kelleher 3rd, R.J. Flanagan P.M. Kornberg R.D. Horikoshi M. Roeder R.G. Nature. 1994; 369: 252-255Crossref PubMed Scopus (101) Google Scholar). In yeast, different residues Arg-231, Arg-235, and Phe-250 contribute to the TBP surface that interacts with BRF (29Shen Y. Kassavetis G.A. Bryant G.O. Berk A.J. Mol. Cell. Biol. 1998; 18: 1692-1700Crossref PubMed Scopus (43) Google Scholar). In our assays, single-amino acid substitutions R235E and F250E in TBP prevent BRFU from entering a TBP·DNA complex. Mutation E284R did not affect the stability of the BRFU·TBP·DNA complex but caused significant changes in the complex conformation. Thus, these data are consistent with the BRFU sequence similarities to both BRF and TFIIB. Clearly, the sequences flanking the TATA-box in the U2 TATA construct do not allow TBP·TFIIB·DNA complex formation and thus might exclude TFIIF-pol II recruitment, and in consequence, pol II-specific transcription. However, because BRFU forms a complex with TBP·AdMLP as well as TFIIB does and pol III-type transcription can initiate from an snRNA gene promoter containing an mRNA gene TATA-box and flanking sequences, additional mechanism(s) must direct BRFU to specifically assemble on pol III snRNA templates in the cell. Otherwise, BRFU would compete with TFIIB in PIC formation on the promoters of protein-encoding genes (Fig.7). Thus, sequences further outside the core promoter of the AdMLP may ensure that only pol II-specific PICs can form on this template in vivo. For instance, factors binding to promoter and initiator sequences may interact specifically with pol II-specific basic factors and effectively exclude pol III-specific factors. However, at least one mRNA promoter, within the c-myc gene, can direct TATA-dependent transcription by pol III both in vitro and in vivo in some circumstances (46Sussman D.J. Chung J. Leder P. Nucleic Acids Res. 1991; 19: 5045-5052Crossref PubMed Scopus (18) Google Scholar), suggesting that the balance can be tipped to favor pol III. The availability of individual factors, TBP, PTF, B“, and pol III snRNA-specific BRFU offers a unique system to understand the structure and function of a basal transcription multisubunit complex specific for snRNA genes transcribed by pol III polymerase. It is possible that, in addition to TBP, BRFU or its associated factors directly contact transcription factors PTF and B” and, together with any recognition elements in the core of pol III snRNA promoters, provides a basis for selective recruitment of pol III. We thank Nouria Hernandez for plasmid encoding His-tagged BRFU and Arnold Berk for plasmids encoding AS TBP: wt, R231E, R235E, F250E, and E284R. Human recombinant untagged TBP was a generous gift from Joost Zomerdijk. His-tagged core TBP and AS TBP were kindly prepared by Diana Boyd.
TATA box
TATA-Box Binding Protein
RNA polymerase III
RNA polymerase II
Small nuclear RNA
CAAT box
Transcription
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Citations (28)
TATA-Box Binding Protein
TATA-binding protein
Transcription
TATA box
Cite
Citations (23)