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• W2085350060 abstract "Transcription factor (TF) IIA performs two important regulatory functions during RNA polymerase II transcription: it is required for efficient binding of TFIID to a core promoter and it mediates the effects of upstream activators, both through direct interaction with the TATA box binding protein (TBP). To begin studying how TFIIA mediates these effects, we used a highly sensitive protease footprinting methodology to identify surfaces of human TFIIA participating in TFIIA·TBP·TATA ternary complex formation. Chymotrypsin and proteinase K cleavage patterns of TFIIA bearing a 32P-end-labeled γ subunit revealed that amino acids 59-73 were protected from cleavage both in the context of an immobilized ternary complex and in a binary complex with TBP alone. In contrast, amino acids 341-367 in the β portion of a 32P-labeled α-β subunit were protected in the ternary but not in the binary complex, implying that those residues interact with promoter DNA. The regions of human TFIIA identified by protease footprinting are homologous to and encompass the yeast TFIIA residues that contact TBP and DNA in the recently solved crystal structure of the yeast ternary complex. The conservation of the regions and residues mediating complex formation implies that yeast and human TFIIA employ the same mechanism to stabilize the binding of TFIID to a core promoter. Transcription factor (TF) IIA performs two important regulatory functions during RNA polymerase II transcription: it is required for efficient binding of TFIID to a core promoter and it mediates the effects of upstream activators, both through direct interaction with the TATA box binding protein (TBP). To begin studying how TFIIA mediates these effects, we used a highly sensitive protease footprinting methodology to identify surfaces of human TFIIA participating in TFIIA·TBP·TATA ternary complex formation. Chymotrypsin and proteinase K cleavage patterns of TFIIA bearing a 32P-end-labeled γ subunit revealed that amino acids 59-73 were protected from cleavage both in the context of an immobilized ternary complex and in a binary complex with TBP alone. In contrast, amino acids 341-367 in the β portion of a 32P-labeled α-β subunit were protected in the ternary but not in the binary complex, implying that those residues interact with promoter DNA. The regions of human TFIIA identified by protease footprinting are homologous to and encompass the yeast TFIIA residues that contact TBP and DNA in the recently solved crystal structure of the yeast ternary complex. The conservation of the regions and residues mediating complex formation implies that yeast and human TFIIA employ the same mechanism to stabilize the binding of TFIID to a core promoter. INTRODUCTIONTranscription of protein-encoding genes by RNA polymerase II (pol II) 1The abbreviations used are: pol IIRNA polymerase IITFtranscription factorTBPTATA box binding proteinTAFTBP-associated factorUSAUpstream Factor Stimulatory Activitytstemperature-sensitiveHMKheart muscle kinaseGSTglutathione-S-transferasePMSFphenylmethylsulfonyl fluoride, DTT, dithiothreitol. is regulated by an intricate array of protein-protein and protein-DNA interactions (1Tjian R. Maniatis T. Cell. 1994; 77: 5-8Google Scholar, 2Zawel L. Reinberg D. Annu. Rev. Biochem. 1995; 64: 533-561Google Scholar, 3Hori R. Carey M. Curr. Opin. Genet. & Dev. 1994; 4: 236-244Google Scholar, 4Conaway R.C. Conaway J.W. Annu. Rev. Biochem. 1993; 62: 161-190Google Scholar). Understanding how these interactions mediate formation of a transcription complex over a core promoter is a central problem in the field of gene expression. In the step-wise model, transcription complex assembly is nucleated by binding of the general initiation factors (Transcription Factor) TFIID and TFIIA to the TATA box generating the “DA complex” (5Zawel L. Reinberg D. Prog. Nucleic Acid Res. Mol. Biol. 1993; 44: 67-108Google Scholar). The parallel between the ability of gene activators to facilitate DA complex formation and to activate transcription suggests that the complex plays a key role in regulation (6Chi T. Lieberman P. Ellwood K. Carey M. Nature. 1995; 377: 254-257Google Scholar).TFIID is a multisubunit complex consisting of TATA box binding protein (TBP) and eight or more TBP-associated factors (TAFs) (7Dynlacht B.D. Hoey T. Tjian R. Cell. 1991; 66: 563-576Google Scholar, 8Zhou Q. Lieberman P.M. Boyer T.G. Berk A.J. Genes & Dev. 1992; 6: 1964-1974Google Scholar, 9Poon D. Bai Y. Campbell A.M. Bjorklund S. Kim Y.J. Zhou S. Kornberg R.D. Weil P.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8224-8228Google Scholar, 10Reese J.C. Apone L. Walker S.S. Griffin L.A. Green M.R. Nature. 1994; 371: 523-527Google Scholar). TBP alone, when it is substituted for TFIID, can nucleate the formation of a basal transcription complex that is fully functional but not responsive to activators. In contrast, TAFs are thought to act as co-activators because they are dispensable for basal transcription but are required to obtain activator-responsive transcription in vitro. In addition to TAFs, a fraction called upstream-factor stimulatory activity (USA), which contains both positive and negative co-regulators, potentiates transcriptional activation (11Meisterernst M. Roy A.L. Lieu H.M. Roeder R.G. Cell. 1991; 66: 981-993Google Scholar).In the DA complex, TFIIA functions both to stabilize the relatively weak binding between TFIID and the TATA box and is necessary for activator recruitment of TFIID (12Lieberman P.M. Berk A.J. Genes & Dev. 1994; 8: 995-1006Google Scholar, 13Kobayashi N. Boyer T.G. Berk A.J. Mol. Cell. Biol. 1995; 15: 6465-6473Google Scholar, 14Shykind B.M. Kim J. Sharp P.A. Genes & Dev. 1995; 9: 1354-1365Google Scholar); its role in recruitment depends on the TAFs since activators have no effect on TBP·TFIIA complex formation. In the stepwise model, the formation of the transcription complex is completed by the successive association of TFIIB, RNA polymerase/TFIIF, TFIIE, and TFIIH (2Zawel L. Reinberg D. Annu. Rev. Biochem. 1995; 64: 533-561Google Scholar, 4Conaway R.C. Conaway J.W. Annu. Rev. Biochem. 1993; 62: 161-190Google Scholar). An alternative model for complex formation involves a holoenzyme (15Koleske A.J. Young R.A. Trends Biochem. Sci. 1995; 20: 113-116Google Scholar) containing RNA polymerase II and many of the other general initiation factors. The holoenzyme was discovered initially in yeast and more recently in mammalian cells. Both the yeast and some mammalian versions have been reported to lack TFIID and TFIIA (16Chao D.M. Gadbois E.L. Murray P.J. Anderson S.F. Sonu M.S. Parvin J.D. Young R.A. Nature. 1996; 380: 82-85Google Scholar, 17Koleske A.J. Young R.A. Nature. 1994; 368: 466-469Google Scholar, 18Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Google Scholar). One possible function of the DA subcomplex is to form an activator-responsive platform for recruitment of the holoenzyme.Human (and Drosophila) TFIIA is a multisubunit protein consisting of three subunits called α (LN), β (LC), and γ (S) (19Ma D. Watanabe H. Mermelstein F. Admon A. Oguri K. Sun X. Wada T. Imai T. Shiroya T. Reinberg D. Handa H. Genes & Dev. 1993; 7: 2246-2257Google Scholar, 20Ozer J. Moore P.A. Bolden A.H. Lee A. Rosen C.A. Lieberman P.M. Genes & Dev. 1994; 8: 2324-2335Google Scholar, 21Sun X. Ma D. Sheldon M. Yeung K. Reinberg D. Genes & Dev. 1994; 8: 2336-2348Google Scholar, 22DeJong J. Roeder R.G. Genes & Dev. 1993; 7: 2220-2234Google Scholar, 23Yokomori K. Zeidler M.P. Chen J.L. Verrijzer C.P. Mlodzik M. Tjian R. Genes & Dev. 1994; 8: 2313-2323Google Scholar, 24Yokomori K. Admon A. Goodrich J.A. Chen J.L. Tjian R. Genes & Dev. 1993; 7: 2235-2245Google Scholar, 25Cortes P. Flores O. Reinberg D. Mol. Cell. Biol. 1992; 12: 413-421Google Scholar). α and β are synthesized as a precursor that is processed proteolytically to generate the mature subunits although the unprocessed form is functional in vitro. Yeast TFIIA consists of only two subunits, TOA1 and TOA2 (26Ranish J.A. Lane W.S. Hahn S. Science. 1992; 255: 1127-1129Google Scholar, 27Ranish J.A. Hahn S. J. Biol. Chem. 1991; 266: 19320-19327Google Scholar). TOA1 is homologous at its amino and carboxyl termini to regions of the human α and β subunits, respectively, and TOA2 is homologous to the human γ subunit (19Ma D. Watanabe H. Mermelstein F. Admon A. Oguri K. Sun X. Wada T. Imai T. Shiroya T. Reinberg D. Handa H. Genes & Dev. 1993; 7: 2246-2257Google Scholar, 20Ozer J. Moore P.A. Bolden A.H. Lee A. Rosen C.A. Lieberman P.M. Genes & Dev. 1994; 8: 2324-2335Google Scholar, 21Sun X. Ma D. Sheldon M. Yeung K. Reinberg D. Genes & Dev. 1994; 8: 2336-2348Google Scholar, 22DeJong J. Roeder R.G. Genes & Dev. 1993; 7: 2220-2234Google Scholar). Analysis of systematic internal deletion mutants of both subunits demonstrated that all of the regions conserved between yeast and human TFIIA were required for yeast viability (28Kang J.J. Auble D.T. Ranish J.A. Hahn S. Mol. Cell. Biol. 1995; 15: 1234-1243Google Scholar). For TOA1 (α and β), these deletion mutants defined the amino- and carboxyl-terminal ends as essential but the nonconserved, middle region as dispensable for viability. A deletion mutant removing residues 217-240 abolishes binding to TBP. For TOA2 (γ), all of the internal deletions but one resulted in inviable yeast, making it difficult to genetically define functional domains. Alanine scanning mutants of both subunits were screened for temperature-sensitive (ts) growth phenotypes. In TOA1, all the ts mutations were in basic residues between residues 253 and 259. These mutants bound normally to TBP but could not form complexes, implying a decreased ability to bind DNA. Among the three ts mutants identified in TOA2, one double mutant, D73A/D74A, bound less tightly to TBP although complex formation was unaffected, and the other two exhibited no detectable difference in their ability to bind TBP or form ternary complexes. Human TFIIAγ has also been analyzed by alanine substitution mutations and Y65A (equals Y69 in yeast TOA2) caused the greatest decrease in the ability of IIA to stabilize TBP binding (29Ozer J. Bolden A.H. Lieberman P.M. J. Biol. Chem. 1996; 271: 11182-11190Google Scholar).The crystal structure of the ternary complex containing the yeast (y)CYC1 TATA box, yTBP, and yTFIIA was recently published (30Tan S. Hunziker Y. Sargent D.F. Richmond T.J. Nature. 1996; 381: 127-134Google Scholar, 31Geiger J.H. Hahn S. Lee S. Sigler P.B. Science. 1996; 272: 830-836Google Scholar). In the crystal structure, yTBP, which encodes two 80-amino acid direct repeats, folds into a pseudosymmetric saddle-like shape containing a hydrophobic concave surface composed of a 10-strand antiparallel β-sheet, which interacts with the minor groove of the DNA. TBP binding distorts the TATA box by inserting phenylalanine residues between the first and final base pairs of the recognition site, resulting in bending of the DNA by 80° and broadening of the minor groove (32Kim Y. Geiger J.H. Hahn S. Sigler P.B. Nature. 1993; 365: 512-520Google Scholar, 33Kim J.L. Nikolov D.B. Burley S.K. Nature. 1993; 365: 520-527Google Scholar). yTFIIA, on the other hand, is composed of a four-helix bundle and a 12-strand β-barrel (30Tan S. Hunziker Y. Sargent D.F. Richmond T.J. Nature. 1996; 381: 127-134Google Scholar, 31Geiger J.H. Hahn S. Lee S. Sigler P.B. Science. 1996; 272: 830-836Google Scholar). The β-barrel is made of six strands each from the carboxyl terminus of TOA1 (TOA1C∼human TFIIAβ) and the carboxyl terminus of TOA2 (γ). The four-helix bundle is composed of the amino terminus of TOA1 (TOA1N∼human TFIIAα) and the amino terminus of TOA2. The interaction between TFIIA and TBP places the β-sheets of TOA2 and TBP in close proximity, resulting in a continuous 16-strand β-sheet. TFIIA interacts with DNA through basic residues in TOA1C that lie within a loop connecting two β-strands.DNA-protein cross-linking studies have confirmed certain aspects of the structure in solution (34Coulombe B. Li J. Greenblatt J. J. Biol. Chem. 1994; 269: 19962-19967Google Scholar, 35Legrange T. Kim T.K. Orphanides G. Ebright Y. Ebright R. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10620-10625Google Scholar). Collectively, these studies show cross-linking was observed between DNA and α upstream of the TATA box, between β and both sides of the TATA box, and with γ at one position upstream of the TATA. In the crystal structure of the yeast complex, only a region homologous to part of the β subunit contacts DNA.To study how TFIIA stabilizes TFIID/TBP binding, we investigated the assembly of the ternary complex containing a mammalian TATA box, human TBP, and human TFIIA. Presently, there is no genetic analysis of the α-β subunit of human TFIIA or crystal structure of free TFIIA. To identify the interactions mediating complex formation, the differences between free TFIIA and complexed TFIIA were characterized using protease footprinting. The interactions of TFIIA with TBP and DNA were identified along with the accessible regions of TFIIA. The results of our protease footprinting studies are placed within the context of the recently solved x-ray structure of the yeast ternary complex.DISCUSSIONWe have identified, using protease footprinting, interactions made by human TFIIA upon assembly of the TFIIA-TBP-TATA box ternary complex. Each subunit makes a series of contacts with one of the other components of the complex, the TFIIAγ subunit with TBP and the IIAα-β subunit with DNA. These two interactions allow TFIIA to form a bracket that stabilizes TBP binding. These results are summarized schematically in Fig. 4. Together, TBP and TFIIA function to provide specificity and stability. The preference of TBP for the TATA box provides the specificity required to bring TFIID to the promoter, but the weak affinity of TFIIA for DNA is used advantageously to stabilize binding of TFIID to promoters, which contain diverse sequences flanking the TATA box.Correlations with Crystal Structure and Biochemical DataThe contacts made by TFIIA within the human ternary complex as measured by protease footprinting are similar to those made within the yeast ternary complex as identified in the crystal structure. Fig. 5A is a ribbon diagram illustrating the recently solved crystal structure of the yeast ternary complex (30Tan S. Hunziker Y. Sargent D.F. Richmond T.J. Nature. 1996; 381: 127-134Google Scholar, 31Geiger J.H. Hahn S. Lee S. Sigler P.B. Science. 1996; 272: 830-836Google Scholar) in which yTOA1 (α and β), yTOA2 (γ), TBP, and the TATA box oligonucleotide are yellow, green, blue, and white, respectively. In the structure, the two subunits of yTFIIA are highly intertwined and form a boot-shaped structure in which TOA2 (γ) interacts with TBP and TOA1 (α and β) interacts with DNA. TFIIA binds to the underside of TBP along the loop (stirrup) (42Nikolov D.B. Hu S.H. Lin J. Gasch A. Hoffmann A. Horikoshi M. Chua N.H. Roeder R.G. Burley S.K. Nature. 1992; 360: 40-46Google Scholar) connecting β-strand 2 and β-strand 3. A mutagenesis study, in which radical changes were introduced into surface residues of human TBP, identified an interaction between TFIIA and residues encompassing the stirrup within the first direct repeat of TBP, consistent with the crystal structure (43Bryant G.O. Martel L.S. Burley S.K. Berk A.J. Genes & Dev. 1996; 10: 2491-2504Google Scholar). The ability of these TBP mutants to support transcriptional activation in vivo and TBP-TFIIA complex formation in vitro was greatly diminished.Fig. 5Summary of results modeled on the crystal structure. A-D, ribbon model of the yTFIIA-yTBP-TATA box ternary complex. Yeast TFIIA (homologous to TFIIAα-β), yTOA2 (homologous to TFIIAγ), yTBP, and DNA containing a TATA box are yellow, green, light blue, and white, respectively. The view in panel A is from the same angle used in panels B and C. The view in panel D is the same one used in panels E and F. B, space-filling model of yTFIIA. Yeast TFIIA (homologous to TFIIAα-β) and yTOA2 (homologous to TFIIAγ) are yellow and green, respectively. The residues of hTFIIA (i.e. the homologous ones in yTOA2) identified by protease footprinting as interacting with TBP are identified as red. C, space-filling model of the ternary complex. yTFIIA is shown from the same view as in panel B and using the same color scheme. yTBP and DNA containing a TATA box are light blue and white, respectively. The interaction between the red residues of the γ subunit and TBP can be easily visualized. E, space-filling model of yTFIIA. The color scheme is the same as in panel B except that the residues of hTFIIA (i.e. the homologous ones in yTOA1) identified by protease footprinting as interacting with DNA are identified as red. F, space-filling model of the ternary complex. yTFIIA is shown from the same view as in panel E and using the same color scheme. yTBP and DNA containing a TATA box are light blue and white, respectively. The interaction between the red residues of the α-β subunit and DNA can be easily visualized.View Large Image Figure ViewerDownload (PPT)In Figs. 5B, C, E, and F, our footprinting results are superimposed on space-filling models of yeast TFIIA (Fig. 5, B and E) alone and in the ternary complex (Fig. 5, C and F). Fig. 5, B and C, shows the γ-TBP interaction using the same view as in Fig. 5A. Fig. 5B illustrates yTFIIA alone in which TOA1 (i.e. hTFIIAα-β) is yellow and TOA2 (i.e. hTFIIAγ) is green. The residues identified by protease footprinting as interacting with TBP (hTFIIAγ residues 59-73 homologous to yTOA2 residues 63-77) are colored red. Of the 15 residues in the protected region, 11 of the positions are homologous between human and yeast including all those that directly contact TBP.In Fig. 5C, TBP (blue) and the TATA box oligonucleotide (white) have been added to TFIIA. The protected region in red forms β-strands that are part of the domain that is intertwined with the β-strands of yTOA1 to form the β-barrel. A comparison of Fig. 5B with 5C illustrates that the edge of the concave β-sheet region of TBP covers the “red” region and explains why TBP protects that region from proteolysis.Fig. 5, D-F, is a separate view of the ternary complex used to optimize presentation of the region of hTFIIAα-β protected in the ternary complex. Fig. 5, E and F, superimposes the interaction between TFIIAα-β and DNA identified by protease footprinting onto TFIIA alone (Fig. 5E) and in the ternary complex (Fig. 5F). In this case, the residues protected in hTFIIAα-β (TOA2) are labeled as red, and this region lies within the β-strands of the β-portion of hTFIIAα-β. Within the identified region, 16 of the 27 residues are homologous between yeast and human, including 6 basic ones.The region of the human TFIIAα-β subunit protected from proteolysis in the ternary complex includes the homologous residues of yeast TOA1 shown to contact DNA directly. In Fig. 5F, when yTBP (blue) and the TATA box oligonucleotide (white) are added to yTFIIA (shown from the same view and with the same color scheme in Fig. 5E), the DNA runs across the “red” residues, which is consistent with the observed protease footprint. It appears from the figure that the protected residue at the bottom of the figure may still be accessible from underneath. However, the TATA box oligonucleotide used for our footprinting experiments is longer than the one used in the crystallography studies, which would increase the protection of that site.The one interaction observed in the crystal structure that was not detected in our study was the insertion of the penultimate tryptophan residue of yTOA1 into a crevice created where TBP and yTOA2 interact. Because that tryptophan is the carboxyl-terminal residue in hTFIIAα-β, it is probably too close to our HMK tag for detection.Although the protease footprinting analysis does not identify other interactions of hTFIIA within the ternary complex, it is possible that a protease-resistant region forms other contacts. The lack of a proteolytic reagent that cleaves at every surface position does tend to limit the protease footprinting approach. One possible limitation is revealed by mutational studies that indicate that an interaction exists between a highly acidic region of hTFIIA α-β and the H2 helix of TBP (44Buratowski S. Zhou H. Science. 1992; 255: 1130-1132Google Scholar, 45Lee D.K. DeJong J. Hashimoto S. Horikoshi M. Roeder R.G. Mol. Cell. Biol. 1992; 12: 5189-5196Google Scholar, 46Tang H. Sun X. Reinberg D. Ebright R.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1119-1124Google Scholar). We have not identified such a region in our study.Interactions of TFIIA with TBP in the absence of DNA have been studied using “GST-pulldown” experiments. Interactions of the α, β, and γ subunits (or their homologues) individually with TBP have been identified. However, in view of the crystal structure, the previous studies will have to be reevaluated because the subunits are intertwined and probably will not fold properly on their own. Our studies define an interaction between only the γ subunit and TBP.A protein-DNA cross-linking study concluded the α subunit interacts with promoter DNA upstream of TBP centered at −45, the γ subunit is close to only −39, and the β subunit can be cross-linked to both sides of TBP (35Legrange T. Kim T.K. Orphanides G. Ebright Y. Ebright R. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10620-10625Google Scholar). These data are similar to an earlier study (34Coulombe B. Li J. Greenblatt J. J. Biol. Chem. 1994; 269: 19962-19967Google Scholar). We detect only an interaction between the β portion of the α-β subunit and DNA. The absence of an α-DNA interaction may simply reflect the use of a TATA box oligonucleotide that is too short to detect this interaction. It may also be possible that some proportion of TFIIA undergoes a conformational change within the ternary complex leading to these other DNA contacts, a finding supported by at least one recent study (21Sun X. Ma D. Sheldon M. Yeung K. Reinberg D. Genes & Dev. 1994; 8: 2336-2348Google Scholar).Based on protease sensitivity, it is plausible that other proteins will interact with TFIIA via the carboxyl terminus of the γ subunit and with the α subunit. Because the γ subunit binds TBP, regulatory proteins interacting with the γ subunit could modulate the ability of TFIIA to associate with TBP. There is evidence that the Epstein-Barr virus activator ZEBRA overcomes a mutation in the γ subunit, which decreases TBP binding and complex formation (29Ozer J. Bolden A.H. Lieberman P.M. J. Biol. Chem. 1996; 271: 11182-11190Google Scholar). Because the α subunit is upstream within the ternary complex (30Tan S. Hunziker Y. Sargent D.F. Richmond T.J. Nature. 1996; 381: 127-134Google Scholar, 31Geiger J.H. Hahn S. Lee S. Sigler P.B. Science. 1996; 272: 830-836Google Scholar), α is positioned to more easily interact with activators or possibly TAFs that extend toward upstream bound activators.Our understanding of how the transcription complex is assembled requires an understanding of each step, particularly the key regulatory ones. We have characterized the formation of a promoter-bound complex containing TBP and TFIIA. We will use this study as a starting point to perform parallel studies with TFIID and to examine the interactions of activators. It will be particularly interesting to determine how (and if) TAFs change the interaction of IIA with TBP. The ability of activators to both facilitate complex assembly and induce a conformational change in the DA complex could be studied. These future studies will benefit from using smaller proteolytic reagents, especially given the larger size of TFIID relative to TBP. Recent experiments by the labs of Meares, Heyduk, and Ebright and coworkers (47Greiner D.P. Hughes K.A. Gunasekera A.H. Meares C.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 71-75Google Scholar, 48Heyduk E. Heyduk T. Biochemistry. 1994; 33: 9643-9650Google Scholar, 49Heyduk T. Heyduk E. Severinov K. Tang H. Ebright R.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10162-10166Google Scholar) have shown that hydroxyl-radicals can be an effective reagent for protease footprinting studies and will probably be a useful reagent for our studies. INTRODUCTIONTranscription of protein-encoding genes by RNA polymerase II (pol II) 1The abbreviations used are: pol IIRNA polymerase IITFtranscription factorTBPTATA box binding proteinTAFTBP-associated factorUSAUpstream Factor Stimulatory Activitytstemperature-sensitiveHMKheart muscle kinaseGSTglutathione-S-transferasePMSFphenylmethylsulfonyl fluoride, DTT, dithiothreitol. is regulated by an intricate array of protein-protein and protein-DNA interactions (1Tjian R. Maniatis T. Cell. 1994; 77: 5-8Google Scholar, 2Zawel L. Reinberg D. Annu. Rev. Biochem. 1995; 64: 533-561Google Scholar, 3Hori R. Carey M. Curr. Opin. Genet. & Dev. 1994; 4: 236-244Google Scholar, 4Conaway R.C. Conaway J.W. Annu. Rev. Biochem. 1993; 62: 161-190Google Scholar). Understanding how these interactions mediate formation of a transcription complex over a core promoter is a central problem in the field of gene expression. In the step-wise model, transcription complex assembly is nucleated by binding of the general initiation factors (Transcription Factor) TFIID and TFIIA to the TATA box generating the “DA complex” (5Zawel L. Reinberg D. Prog. Nucleic Acid Res. Mol. Biol. 1993; 44: 67-108Google Scholar). The parallel between the ability of gene activators to facilitate DA complex formation and to activate transcription suggests that the complex plays a key role in regulation (6Chi T. Lieberman P. Ellwood K. Carey M. Nature. 1995; 377: 254-257Google Scholar).TFIID is a multisubunit complex consisting of TATA box binding protein (TBP) and eight or more TBP-associated factors (TAFs) (7Dynlacht B.D. Hoey T. Tjian R. Cell. 1991; 66: 563-576Google Scholar, 8Zhou Q. Lieberman P.M. Boyer T.G. Berk A.J. Genes & Dev. 1992; 6: 1964-1974Google Scholar, 9Poon D. Bai Y. Campbell A.M. Bjorklund S. Kim Y.J. Zhou S. Kornberg R.D. Weil P.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8224-8228Google Scholar, 10Reese J.C. Apone L. Walker S.S. Griffin L.A. Green M.R. Nature. 1994; 371: 523-527Google Scholar). TBP alone, when it is substituted for TFIID, can nucleate the formation of a basal transcription complex that is fully functional but not responsive to activators. In contrast, TAFs are thought to act as co-activators because they are dispensable for basal transcription but are required to obtain activator-responsive transcription in vitro. In addition to TAFs, a fraction called upstream-factor stimulatory activity (USA), which contains both positive and negative co-regulators, potentiates transcriptional activation (11Meisterernst M. Roy A.L. Lieu H.M. Roeder R.G. Cell. 1991; 66: 981-993Google Scholar).In the DA complex, TFIIA functions both to stabilize the relatively weak binding between TFIID and the TATA box and is necessary for activator recruitment of TFIID (12Lieberman P.M. Berk A.J. Genes & Dev. 1994; 8: 995-1006Google Scholar, 13Kobayashi N. Boyer T.G. Berk A.J. Mol. Cell. Biol. 1995; 15: 6465-6473Google Scholar, 14Shykind B.M. Kim J. Sharp P.A. Genes & Dev. 1995; 9: 1354-1365Google Scholar); its role in recruitment depends on the TAFs since activators have no effect on TBP·TFIIA complex formation. In the stepwise model, the formation of the transcription complex is completed by the successive association of TFIIB, RNA polymerase/TFIIF, TFIIE, and TFIIH (2Zawel L. Reinberg D. Annu. Rev. Biochem. 1995; 64: 533-561Google Scholar, 4Conaway R.C. Conaway J.W. Annu. Rev. Biochem. 1993; 62: 161-190Google Scholar). An alternative model for complex formation involves a holoenzyme (15Koleske A.J. Young R.A. Trends Biochem. Sci. 1995; 20: 113-116Google Scholar) containing RNA polymerase II and many of the other general initiation factors. The holoenzyme was discovered initially in yeast and more recently in mammalian cells. Both the yeast and some mammalian versions have been reported to lack TFIID and TFIIA (16Chao D.M. Gadbois E.L. Murray P.J. Anderson S.F. Sonu M.S. Parvin J.D. Young R.A. Nature. 1996; 380: 82-85Google Scholar, 17Koleske A.J. Young R.A. Nature. 1994; 368: 466-469Google Scholar, 18Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Google Scholar). One possible function of the DA subcomplex is to form an activator-responsive platform for recruitment of the holoenzyme.Human (and Drosophila) TFIIA is a multisubunit protein consisting of three subunits called α (LN), β (LC), and γ (S) (19Ma D. Watanabe H. Mermelstein F. Admon A. Oguri K. Sun X. Wada T. Imai T. Shiroya T. Reinberg D. Handa H. Genes & Dev. 1993; 7: 2246-2257Google Scholar, 20Ozer J. Moore P.A. Bolden A.H. Lee A. Rosen C.A. Lieberman P.M. Genes & Dev. 1994; 8: 2324-2335Google Scholar, 21Sun X. Ma D. Sheldon M. Yeung K. Reinberg D. Genes & Dev. 1994; 8: 2336-2348Google Scholar, 22DeJong J. Roeder R.G. Genes & Dev. 1993; 7: 2220-2234Google Scholar, 23Yokomori K. Zeidler M.P. Chen J.L. Verrijzer C.P. Mlodzik M. Tjian R. Genes & Dev. 1994; 8: 2313-2323Google Scholar, 24Yokomori K. Admon A. Goodrich J.A. Chen J.L. Tjian R. Genes & Dev. 1993; 7: 2235-2245Google Scholar, 25Cortes P. Flores O. Reinberg D. Mol. Cell. Biol. 1992; 12: 413-421Google Scholar). α and β are synthesized as a precursor that is processed proteolytically to generate the mature subunits although the unprocessed form is functional in vitro. Yeast TFIIA consists of only two subunits, TOA1 and TOA2 (26Ranish J.A. Lane W.S. Hahn S. Science. 1992; 255: 1127-1129Google Scholar, 27Ranish J.A. Hahn S. J. Biol. Chem. 1991; 266: 19320-19327Google Scholar). TOA1 is homologous at its amino and carboxyl termini to regions of the human α and β subunits, respectively, and TOA2 is homologous to the human γ subunit (19Ma D. Watanabe H. Mermelstein F. Admon A. Oguri K. Sun X. Wada T. Imai T. Shiroya T. Reinberg D. Handa H. Genes & Dev. 1993; 7: 2246-2257Google Scholar, 20Ozer J. Moore P.A. Bolden A.H. Lee A. Rosen C.A. Lieberman P.M. Genes & Dev. 1994; 8: 2324-2335Google Scholar, 21Sun X. Ma D. Sheldon M. Yeung K. Reinberg D. Genes & Dev. 1994; 8: 2336-2348Google Scholar, 22DeJong J. Roeder R.G. Genes & Dev. 1993; 7: 2220-2234Google Scholar). Analysis of systematic internal deletion mutants of both subunits demonstrated that all of the regions conserved between yeast and human TFIIA were required for yeast viability (28Kang J.J. Auble D.T. Ranish J.A. Hahn S. Mol. Cell. Biol. 1995; 15: 1234-1243Google Scholar). For TOA1 (α and β), these deletion mutants defined the amino- and carboxyl-terminal ends as essential but the nonconserved, middle region as dispensable for viability. A deletion mutant removing residues 217-240 abolishes binding to TBP. For TOA2 (γ), all of the internal deletions but one resulted in inviable yeast, making it difficult to genetically define functional domains. Alanine scanning mutants of both subunits were screened for temperature-sensitive (ts) growth phenotypes. In TOA1, all the ts mutations were in basic residues between residues 253 and 259. These mutants bound normally to TBP but could not form complexes, implying a decreased ability to bind DNA. Among the three ts mutants identified in TOA2, one double mutant, D73A/D74A, bound less tightly to TBP although complex formation was unaffected, and the other two exhibited no detectable difference in their ability to bind TBP or form ternary complexes. Human TFIIAγ has also been analyzed by alanine substitution mutations and Y65A (equals Y69 in yeast TOA2) caused the greatest decrease in the ability of IIA to stabilize TBP binding (29Ozer J. Bolden A.H. Lieberman P.M. J. Biol. Chem. 1996; 271: 11182-11190Google Scholar).The crystal structure of the ternary complex containing the yeast (y)CYC1 TATA box, yTBP, and yTFIIA was recently published (30Tan S. Hunziker Y. Sargent D.F. Richmond T.J. Nature. 1996; 381: 127-134Google Scholar, 31Geiger J.H. Hahn S. Lee S. Sigler P.B. Science. 1996; 272: 830-836Google Scholar). In the crystal structure, yTBP, which encodes two 80-amino acid direct repeats, folds into a pseudosymmetric saddle-like shape containing a hydrophobic concave surface composed of a 10-strand antiparallel β-sheet, which interacts with the minor groove of the DNA. TBP binding distorts the TATA box by inserting phenylalanine residues between the first and final base pairs of the recognition site, resulting in bending of the DNA by 80° and broadening of the minor groove (32Kim Y. Geiger J.H. Hahn S. Sigler P.B. Nature. 1993; 365: 512-520Google Scholar, 33Kim J.L. Nikolov D.B. Burley S.K. Nature. 1993; 365: 520-527Google Scholar). yTFIIA, on the other hand, is composed of a four-helix bundle and a 12-strand β-barrel (30Tan S. Hunziker Y. Sargent D.F. Richmond T.J. Nature. 1996; 381: 127-134Google Scholar, 31Geiger J.H. Hahn S. Lee S. Sigler P.B. Science. 1996; 272: 830-836Google Scholar). The β-barrel is made of six strands each from the carboxyl terminus of TOA1 (TOA1C∼human TFIIAβ) and the carboxyl terminus of TOA2 (γ). The four-helix bundle is composed of the amino terminus of TOA1 (TOA1N∼human TFIIAα) and the amino terminus of TOA2. The interaction between TFIIA and TBP places the β-sheets of TOA2 and TBP in close proximity, resulting in a continuous 16-strand β-sheet. TFIIA interacts with DNA through basic residues in TOA1C that lie within a loop connecting two β-strands.DNA-protein cross-linking studies have confirmed certain aspects of the structure in solution (34Coulombe B. Li J. Greenblatt J. J. Biol. Chem. 1994; 269: 19962-19967Google Scholar, 35Legrange T. Kim T.K. Orphanides G. Ebright Y. Ebright R. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10620-10625Google Scholar). Collectively, these studies show cross-linking was observed between DNA and α upstream of the TATA box, between β and both sides of the TATA box, and with γ at one position upstream of the TATA. In the crystal structure of the yeast complex, only a region homologous to part of the β subunit contacts DNA.To study how TFIIA stabilizes TFIID/TBP binding, we investigated the assembly of the ternary complex containing a mammalian TATA box, human TBP, and human TFIIA. Presently, there is no genetic analysis of the α-β subunit of human TFIIA or crystal structure of free TFIIA. To identify the interactions mediating complex formation, the differences between free TFIIA and complexed TFIIA were characterized using protease footprinting. The interactions of TFIIA with TBP and DNA were identified along with the accessible regions of TFIIA. The results of our protease footprinting studies are placed within the context of the recently solved x-ray structure of the yeast ternary complex." @default.
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• W2085350060 title "Protease Footprinting Analysis of Ternary Complex Formation by Human TFIIA" @default.
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