A NOVEL FUNGAL MULTISUBUNIT PROTEIN COMPLEX CRITICAL FOR EXPRESSION OF FUNGAL PROTEINS

Abstract: Identification of yeast TATA-box-binding protein (TBP) associated factors (TAFs). Use of TBP as a protein-affinity ligand to isolate from yeast a multisubunit complex that is required specifically for activated transcription by RNA polymerase II.

Description: A NOVEL FUNGAL MULTISUBUNIT PROTEIN COMPLEX CRITICAL FOR EXPRESSION OF FUNGAL PROTEINS Field of the Invention This invention pertains to proteins required for activated transcription in yeast and fungi, nucleic acids encoding these proteins, and methods of using these proteins. Background of the Inven. Most fungi φportunistic p ogens, producing serious disease only in compromised individuals. the result of _... aging population and an increase in the number of immunocom used patients, specifically patients with acquired immunodeficiency syndrome (AIDS), patients undergoing cancer, and corticosteroid therapy, as well as in patients undergoing organ transplantation, fungal infections are increasing rapidly. Most infections begin by colonization of either the skin, a mucosal membrane, or the respiratory epithelium issage through the initial surface barrier is accomplished through a mechanical break ... the epithelium or enzymatic degradation or spore dissemination. Most fungi are readily killed by neutrophils and are only opportunists, but some species are resi; * • it to phagocytic killing and may infect otherwise healthy individuals. Fungi par "itize many different tissues. Superficial cause indolent lesions of the skin. SuV eous pathogens cause infection through the skin and spread by subcutaneous or lymphatic routes. Opportunistic fungi such as Aspergilb are widespread in the environment and are present in normal flora and cause disease i. stly in immunocompromised individuals. Systemic fungi are the most virulent and may cause progressive disease leading to deep seated :eral infections in otherwise healthy individuals (see e.g. Sherris Medical Microbiology, Third Edition. Kenneth J. Ryan. ed.. 2 Appleton & Lange, Norwalk, CT, 1994). The major fungal pathogens in North America are Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Candida species and Aspergillus species (Medically Important Fungi, Second Edition, Davise H. Larone, Ed. , American Society for Microbiology, Washington, D.C.). Histoplama capsulatum causes histoplasmosis, which may be chronic or progressive and fatal. It is either a localized or disseminated infection, primarily of the reticuloendothelial system. Coccidiodes immitis causes coccidioidomycosis, a highly infectious disease that is endemic to the southwestern United States and may be a chronic, sometimes fatal infection involving the skin, bone, joints, lymph nodes, adrenal glands and central nervous system. Blastomyces dermatitidis causes blastomycosis, a chronic infection characterized by suppurative and granulomatous lesions that begins in the lungs and is disseminated to the skin and bones. Cryptococcus neoformans causes cryptococcosis, which may be a chronic infection involving the central nervous system. Candida albicans is the most frequent cause of candidiasis, which ranges from an acute to a chronic infection involving any part of the body. Aspergillus fiimigatus is one of the most frequent causes of aspergillosis, which is an opportunistic infection in immunosuppressed individuals. Fungi are a distinct class of microorganism, most of which are free-living. They are eukaryotic organisms containing a nuclear membrane, mitochondria and an endoplasmic reticulum. The cell structure includes a rigid cell wall of mannan, glucan, and chitin and a cytoplasmic membrane with a large percentage of ergosterol. The size and morphology of fungi vary. There are monomorphic yeasts and yeast-like organisms including Candida, Cryptococcus, and Saccharomyces. There are monomorphic molds, such as Aspergillus and Coccidiodes. Some thermally dimorphic fungi, such as Blastomyces dermatitidis and Histoplasma capsulatum, grow either in a yeast or mold phase. Only a handful of agents are active against fungi. For life threatening disease caused by any of these fungi, amphotericin B is the agent of choice. This drug, however, is associated with numerous severe side effects such as fever, dyspnea and tachycardia, and dosage is limited over the lifetime of the patient because of renal toxicity . An agent frequently used concurrently is flucytosine, a nucleoside analog that cannot be used independent of other agents because of the rapid appearance of resistance. Untoward effects of treatment with flucytosine include leukopenia, thrombocytopenia, rash, nausea, vomiting, diarrhea, and severe enterocolitis. In conditions where the patient's life is not threatened, ketoconazole can be used as a long-term therapy for blastomycosis, histoplasmosis, or coccidioidomycosis. Fluconazole also has a significant role in the treatment of superficial fungal infections. Both compounds are from the same class, the triazoles, and are cytostatic. The emergence of resistance and hepatic toxicity limit the use of triazoles such as fluconazole and ketoconazole. The newest triazole, itraconazole, has similar pharmacokinetics and spectrum of activity as fluconazole. None of the azoles can be used for life threatening or deep seated fungal infections, but they are effective in reducing colonization of fungi such as Candida and for treating superficial mycoses. All major antifungal agents attack directly or indirectly a component of the cell wall— ergosterol. Amphotericin B and other polyene macrolides interact with ergosterol in the cell membrane and form pores or channels tha* increase the permeability of the membrane. Resistant to amphotericin B in mutant _ jins is accompanied by decreased concentrations of ergosterol in their cell membranes Imidazoles and triazoles inhibit sterol 14-α-demethylase, a microsom d cytochrome • 450-dependent enzyme system. Imidazoles and triazoles thus impair the biosynthesis of ergostero' for the cytoplasmic membrane and lead to the accumulation of 14-α-methyl sterols, whici. impair certain membrane-bound enzyme systems (See e.g. 77ιe Pharmacological Basis of Therapeutics, Eighth Edition, Goodman and Gilman, Pergamon Press, 1990). Development of an effective method and composition for treatment of fungal infections is a critical goal of the pharmaceutical industry. The pharmaceutical industry has made numerous efforts to identify fungal-specific drugs, with only limited success to date. It would be of great value to identify a new class of antifungal drug that blocks a fungal target other than ergosterol. This target should be fungal-specific and should lead to development of a drug that is effective against the organisms that are resistant to current therapy. Drug development often relies o lie screening of a large number of potential inhibitors before a specific lead compound inhibitor is found. Assays developed for such screens are complex and must mimic the physiological activity of t! target protein. Thus, it is critical for the development of these screens to define the proteins involved in the targeted process and to have discovered a means of purifying the necessary components of the assay for use in the assay. In addition, it is useful to have clones for the protein components of the assay to facilitate the production of the components. Therefore, there is a need in the art to identify one or more fungal constituents, preferably polypeptides, that can serve as useful targets for drug intervention, and for methods and compositions for identifying useful anti-fungal agents and treating fungal infections. Summary of the Invention The present invention provides a complex of fungal polypeptides, termed TAFs, that are necessary for activated transcription in fungi such as S. cerevisiae. The complex comprises at least nine associated subunit polypeptides, having molecular masses of about 180 kDa, 145 kDa, 116 kDa, 90 kDa, 68 kDa, 51-54 kDa, 47 kDa, and 30 kDa, respectively. TAF-145, having the sequence set forth in Figure 6B SEQ. I.D. No. 1, binds TATA-box Binding Protein (TBP). The invention also includes nucleic acid sequences encoding TAF-145, as well as DNA vectors and transformed cells suitable for recombinant expression of this polypeptide. In one aspect, the present invention encompasses methods for inhibiting fungal transcription in a fungal cell, comprising contacting the cell with an agent that selectively interferes with the transcriptional activation activity of the yeast TAF complex. In another aspect, the invention provides a method for high-throughput screening of large numbers of test compounds, to identify an agent useful in the treatment of fungal diseases in mammals. The method is carried out by exposing the TAF complex or TAF-145 to TBP in the presence of at least one test compound, followed by identifying those test compounds that inhibit the binding of TBP to the TAF complex or TAF- 145. Brief Description of the Drawings Figure 1 shows transcriptional activation reactions programmed by the DNA template shown in the bottom of the figure. The reactions contained either a yeast whole cell extract (WCE), or the flow-through (FT) or eluate (EL) fractions of column chromatography of the WCE on glutathione-S-transferase (GST) columns or on columns containing TATA-box Binding Protein (TBP) coupled to GST. The GAL 4 activator protein was added as indicated. Figure 2A shows the purification protocol for yeast transactivating factor complex (TAF). Figure 2B shows an SDS-polyacrylamide gel profile of the polypeptides present in the fractions shown in Figure 2A. The migration of molecular mass markers are indicated on the left and the yeast TAF polypeptides are indicated on the right. Figure 2C shows transcription reactions containing increasing amounts of the final hydroxylapatite TAF-containing fraction shown in Figure 2B. Figure 3A shows the polypeptide composition of the final hydroxylapatite fraction of Figure 2B when rechromatographed on a Superose 6 gel filtration column. Figure 3B shows co-immunoprecipitation of yeast TAF polypeptides by an anti-TBP antibody. Figure 3C shows a transcription reaction supplemented with TAF polypeptides immunoprecipitated as in Figure 3B. Figure 4 A shows a protein blot in which TAF polypeptides were resolved by SDS-gel electrophoresis, transferred to nitrocellulose, and incubated with 35 S-labelled yeast (y) or human (h) TBP. Figure 4B shows a similar protein blot as in Figure 4A, except that the indicated C-terminal deletion mutants of TAF- 145 were utilized in parallel with full-length TAF- 145. Figure 5 shows transcription reactions programmed by the RNA polymerase HI template shown at the bottom of the figure. The reactions were carried out by WCE and indicated column fractions as in Figures 1 and 2C. Figure 6A shows the deduced amino acid sequence of yeast TAF-90 SEQ I.D. No. 2 polypeptide and a comparison with the sequence of drosophila TAFII-80 SEQ I.D. No. 3. Figure 6B shows the deduced amino acid sequence of yeast TAF- 145 SEQ. I.D. No. 1 and a comparison with the sequence of drosophila TAFII-250 SEQ I.D. No. 4. Figure 6C shows the disruption of the yeast gene encoding TAF- 0, and Figure 6D shows the disruption of TAF-145 SEQ I.D. No. 1 (bottom). The DNA constructs containing disrupted copies of each gene are shown of each figure. At bottom left are shown Southern blots corifirming correct integration of the disrupted gene within each endogenous gene. At bottom right are shown the growth patterns of spores dissected from each disrupted strain. Figure 6E shows a schematic representation of TAF-containing protein complexes from Drosophila (left) and Saccharomyces (right.) Detailed Description of the Invention All patent applications, patents, and literature references cited in this specification are hereby incorporated by reference in their entirety. In case of conflict, the present description, including definitions, will control. Definitions'. 1. "Basal transcription" refers to transcription activity from an RNA polymerase H-directed promoter in the absence of an upstream transcriptional activator. 2. "Coactivator activity" refers to the activity that allows an upstream transcription factor such as GAL4 or its derivatives to activate transcription from an RNA polymerase H-directed promoter in an in vitro or in vivo reconstituted transcription system. Coactivator activity is further defined as an activity that has no effect on basal transcription. 3. "TATA-box binding protein" or "TBP" is a major component of eukaryotic transcription factors. In higher eukaryotes, TBP is isolated as part of a larger protein complex (see below). 4. "TATA-box binding protein- associated factors" or "TAFs" as used herein refer to polypeptides or complexes of polypeptides required for "coactivator activity " in fungal RNA polymerase II transcription reactions by virtue of their association with TBP. 5. "Functional homology" between TAF polypeptides or complexes of polypeptides indicates that one or more biochemical properties specific to fungal TAFs are shared. Examples of such properties are: the ability to specifically modulate the transcription from RNA polymerase H-directed promoters in the presence of an upstream activator protein, and the capacity to specifically bind TBP as a multisubunit complex or as a single subunit under conditions as described herein. 6. "Sequence homology" is used herein to describe the relatedness of TAFs from different sources. "Substantial" sequence homology means that about 70% , more preferably at least about 80%, and most preferably at least about 90% of the two sequences are identical. The level of sequence homology may also be defined functionally, as in, e.g. the stringency of hybridization conditions under which the two sequences effectively or substantially hybridize. "Stringent" hybridization conditions are defined herein as 0.1X SSC at 65 °C. 7. "TAF subunits" refers to individual polypeptides that comprise the TAF complex. Such polypeptides are distinguished from ar-v polypeptides previously known to be TBP binding proteins. Fungal TAF subunits muj be recombinant or purified from natural sources, and may include stuctural or functional TAF homologues as defined above. 8. A "fungal-specific epitope" of a fungal TAF subunit comprises a three- dimensional structural conformation presented by a folded or assembled TAF polypeptide that is not presented by the homologous mammalian sequence. 8. "Modulating transcription" means altering transcription, and includes increasing or decreasing the rate or level of transcription and changing the responsiveness of transcription to regulatory controls. 9. An "isolated" polypeptide or nucleic acid is defined as one that is unaccompanied by at least some of the material with which it is associated in its natural state. Generally, an isolated polypeptide constitutes jast about 1 %, preferably at least about 10%, and more preferably at least about 50% by weight of the total protein in a given sample. Included in the polypeptide weight are alternative forms such as differentially glycosylated or phosphorylated or otherwise post-tranlationally modified forms. An "isolated" nucleic acid sequence is present as other than a naturally occurring chromosome or transcript in its natural state and typically is removed from at least some of the proteins with which it is normally associated with on a natural chromosome, partially pure nucleotide sequence constitutes at least about 5 % , preferably at least about 30%, and more preferably at least about 90% by weight of total nucleic acid present in a given fraction. The present invention is based on the surprising finding that th east Saccharomyces cerevisiae contains "TATA-box binding protein-associated facti , or TAFs, which function in regulating the activity of RNA Polymerase H. Prior to the work reported herein (see Example 1 below), no biochemical or genetic evidence had indicated the existence of such polypeptides in yeast. The discovery and characterization of these polypeptide factors, and the elucidation of differences between their yeast and mammalian versions, implicate these proteins as important targets for the development of new methods and compositions for treatment of fungal infections. The present invention is thus directed towards selective interference with fungal TAF activity, under conditions in which there is no effect on mammalian TAF function. TAF Complexes. Subunit Polypeptides. and Nucleic Acids The present invention provides yeast TAFs, which comprise a complex of nine polypeptides, or closely related families of polypeptides, having molecular masses of about 180, 145, 116, 90, 68, 62, 51-54, 47, and 30 kDa. The complexes, and polypeptide components thereof, may be isolated by virtue of their affinity to fungal or human TBP (see Example 1 below), by the use of chromatographic procedures that take advantage of physico-chemical characteristics of the complexes or of individual subunits, or by affinity to TAF-specific antibodies. The isolated complexes may contain all, or only a subset, of the total known complement of TAF subunits. TAF multisubunit complexes may also be reconstituted and purified from translation products of subunit genes, or from recombinantly produced TAF subunits. It is also contemplated that additional TAF subunit polypeptides will be identified using methods disclosed herein, and will be used in practicing the present invention. The present invention also encompasses nucleic acid sequences that encode TAF subunits. Methods for determining the relevant nucleic acid sequences are described in Example 1 below, and the deduced amino acid sequences of two TAF subunit genes, i.e. genes encoding the 90 kDa and 145 kDa TAF polypeptides (TAF-90 and TAF-145, respectively), are shown in Figure 6B. The present invention encompasses DNA and RNA sequences, and sense and antisense sequences. TAF-encoding sequences according to the present invention may be modified by transitions, transversions, deletions, insertions, or other modifications such as alternative splicing. The invention also encompasses genomic TAF sequences and TAF gene flanking sequences, including TAF regulatory sequences. Nucleic acid sequences encoding TAF polypeptides may also be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- noncoding regions, and the like. Other useful heterologous sequences are known to those skilled in the art. Furthermore, the nucleic acids can be modified to alter stability, solubility, binding affinity and specificity. For example, TAF encoding sequences can be selectively methylated. The nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like. In general, nucleic acid manipulations according to the present invention use methods that are well known in the art, as disclosed in e.g. Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor), or Current Protocols in Molecular Biology (Eds. Aufubel, Brent, Kingston, More, Feidman, Smith and Stuhl, Greene Publ. Assoc, Wiley-Interscience, NY, NY, 1992). The invention also provides vectors comprising nucleic acids encoding TAF or TAF analogs. A large number of vectors, including plasmid and fungal vectors, have been described for expression in a variety of eukaryotic and prokaryotic hosts. Advantageously, vectors may also include a promotor operably linked to the TAF encoding portion. The encoded TAF may be expressed by using any suitable vectors and host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. The particular choice of vector/host is not critical to the invention. Vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes. The inserted TAF coding sequences may be synthesized, isolated from natural sources, prepared as hybrids, etc. Ligation of the coding sequences to the transcriptional regulatory sequences may be achieved by known methods. Suitable host cells may be tπmsformed/transfected/infected by any suitable method including electroporation, CaCl2 mediated DNA uptake, fungal infection, microinjection, microprojectile, or other established methods. Appropriate host cells included bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells. Of particular interest are E. coli, B. Subtilis, Saccharomvces cerevisiae, SF9 cells, C129 cells, 293 cells, Neurospora, and CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloid and lymphoid cell lines. Preferred replication systems include M13, ColEl, SV40, baculovirus, lambda, adenovirus, and the like. A large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner of manipulation, etc. are known in the art. Under appropriate expression conditions, host cells can be used as a source of recombinantly produced TAFs. Nucleic acids encoding TAF polypeptides may also be introduced into cells by recombination events. For example, such a sequence can be microinjected into a cell, and thereby effect homologous recombination at the site of an endogenous gene encoding TAF, an analog or pseudogene thereof, or a sequence with substantial identity to an TAF- encoding gene. Other recombination-based methods such as nonhomologous recombinations, deletion of endogenous gene by homologous recombination, especially in pluripotent cells, may also be used. TAF-containing complexes, the individual polypeptides contained therein, and the nucleic acids encoding these polypeptides may be derived from S. cerevisiae, or may be fungal, non-S. cerew ' -ifle-derived proteins with substantial functional or sequence homology to S. cerevisiae TAFs. These TAFs may be derived from fungal sources such as Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Candida species, and Aspergillus species such as Aspergillus fumigatus. Preferably the TAFs are derived from other yeast-like organisms such as Candida, and most preferably, C. albicans. Non-S. cerevisiae TAFs may be identified and isolated by methods that are well-known in the art. These include: antibody cross reactivity; PCR amplification from genomic DNA using degenerate oligonucleotide probes derived from the TAF sequences disclosed herein; low-stringency hybridizations using similar S. cerevisiae probes; and, finally, functional cloning, in which a cDNA expression library derived from another species is used to transform and complement an absent or defective TAF function in S. cerevisiae. The present invention encompasses TAF complexes and subunits purified from wild-type and genetically altered strains of S. cerevisiae, as well as TAFs of all fungal origins recombinantly produced in a non-native context. In one embodiment, a baculovirus expression system permits the recombinant TAF to be modified, processed and transported within a eukaryotic system. In another embodiment, assembly of the TAF complex, or binding of preassembled TAF complexes to TBP, is performed in a reconstituted cell-free system using partially purified or substantially purified components. For example, TAF complexes, or components thereof, may be adsorbed to the surface of a microtiter plate, and incubated with radiolabelled TBP protein. Functional binding of TBP to TAF complexes or components will result in the association of detectable radioactivity with the plate. TAF polypeptides isolated from any source can be modified by methods known in the art. For example, TAF subunits may be phosphorylated or dephosphorylated, glycosylated or deglycosylated, and the like. Especially useful are modifications that alter TAF solubility, membrane transportability, stability, and binding specificity and affinity. Some examples include fatty acid-acylation, proteolysis, and mutations in TBP interaction domains that stabilize binding. TAFs may also be modified with a label capable of providing a detectable signal, for example, at a heart muscle kinase labeling site, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent compounds, etc. Such labeled TAFs thereof find use, for example, as probes in expression screening assays for proteins that interact with TAF, or in assays for TAF binding to TBP. Identification of Functionally Important TAF Domains and Binding Partners The polypeptides, protein complexes, and nucleic acids sequences of the present invention find use in the discovery, design, and development of pharmaceutically useful antifungal agents. The following embodiments of the present invention are directed towards elucidating epitopes and interactions of TAFs and TAF subunits that can be selectively interfered with in a therapeutically beneficial manner. In one embodiment, the known sequence of a TAF subunit e.g. TAF-145 is used to design synthetic peptides comprising portions of the sequence. These peptides range from about 15 to about 50 amino acids in length. Peptides under 60 amino acids in length may be synthesized routinely using commercially available automated synthesizers. The peptides are then added to a cell-free assembly reaction containing e.g. immobilized TAF complex and soluble radiolabelled TBP. Determining which synthetic peptides inhibit some interact of one or more TAF subumts e.g. with other TAF subunits or with other factors, using routine experimentation, is used to identify different functional domains or epitopes of TAF subunits. For example, a peptide (derived from e.g. TAF- 145) that is found using the above-described method to inhibit the binding of TBP to TAF complex is likely to represent a region of TAF-145 that interacts directly with TBP. In a similar manner, associational domains of different TAF subunits that are involved in interactions among subunits, or between TAF subunits or complexes and other transcriptional components, may be systematically identified. These peptides may themselves constitute useful therapeutic reagents, or may serve as the basis for design and formulation of pharmacologically active compositions. In another embodiment, important functional domains of TAFs are identified using classical and reverse genetic methods that are well-known in the art. For example, a nested set of deletion mutants can be prepared from any known TAF sequence. In this embodiment, progressively longer ammo-terminal and carboxy-teπninal deletions (such as those shown for TAF- 145 in Example 1) can be engineered in a particular TAF sequence. The resulting set of mutant sequences can be individually expressed in S. cerevisiae strains under conditions in which the wild-type version of the TAF is not expressed. By monitoring the function of each mutant, it is possible to identify different regions of each TAF polypeptide that are critical for function i.e. functional domains or epitopes. Based on such studies, using methods that are well-known in the art, it is then possible to selectively introduce defined mutations into different regions of the polypeptide, and perform a similar functional analysis. An important aspect of the present invention is the selection of functional domains or epitopes of yeast TAF subunits that are, in addition, structurally or functionally distinct from their mammlian homologues. Such domains are particularly useful as targets for antifungal drugs. In the case of TAF- 145, the yeast version differs in several important respects from its human homologue, TAF-250. Yeast TAF- 145 is approximately half the size of human TAF-250, and the homologous regions display an amino acid similarity and identity of only 58% and 33%, respectively. Yeast TAF-145 lacks the carboxy terminal half of its human counterpart that contains the proposed "Bromo domains" and a region rich in acidic amino acid residues. Identification of important structural and functional domains of TAFs according to the present invention enables the design and production of useful TAF- derived nucleic acid and peptide-based compounds. For example, fusion proteins may be produced between an important TAF domain and e.g. an enzymatically active fragment of a DNA endonculease. The resulting fusion protein, which can be produced in a fungal cell following introduction into the cell of the hybrid DNA operably linked to an expression vector, finds use in modulating TAF-dependent gene transcription. Other useful TAF fusion partners include sequences useful for immobilization. For example, sequences derived from glutathione-S-transferase (GST) provide a binding site for immobilized glutathione, and sequences that form an epitope recognized by an available monoclonal antibody (e.g. 12CA5 monoclonal antibody) provide a binding site for the immobilized antibody. In another example, particular serine, threonine, or tyrosine residues in a TAF sequence may be identified as functionally important sites for phosphorylation of TAF. See e.g. methods disclosed in Roberts et al. (1991) Science 253, 1022-1026 and in Wegner et al. (1992) Science 256, 370-373. Phosphorylation of TAF subunits may be involved in modulating the transcription activation activity of Polymerase H transcribed genes. Identification of these residues will enable, first, the radiolabelling of TAF subunits with 7- 32 P-ATP. Furthermore, if phosphorylation of a particular residue is necessary for transcriptional activity, phosphorylation inhibitors may be designed to block activity. The nucleic acids encoding TAF may also be used to identify other nuclear factors that interact with TAF. In this embodiment, a yeast cDNA library containing fusion genes of cDNA joined with DNA encoding the activation domain of a transcription factor (e.g. Gal4) is co-transfected with fusion genes encoding a portion of TAF and the DNA binding domain of a transcription factor. Clones encoding TAF binding proteins are able to complement the transcription factor and are identified through transcription of a reporter gene. See, e.g. Fields and Song (1989) Nature 340: 245-246 and Chien et al. (1991) Proc Natl Acad Sci USA 88: 9578-9582. It is contemplated that these additional binding partners for TAF will provide additional targets for antifungal drug therapy. Anti-TAF Antibodies The present invention encompasses antibodies that are specific for TAF complexes or subunits identified as described above. The antibodies may be polyclonal or monclonal, and may distinguish TAFs from other nuclear proteins, discriminate TAFs from different species, identify associational or other functional domains, and the like. Such antibodies are conveniently made using the methods and compositions disclosed in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, other references cited herein, as well as immunological and hybridoma technologies known to those in the art. Where natural or synthetic TAF-derived peptides are used to induce an TAF-specific immune response, the peptides may be conveniently coupled to an suitable carrier such as KLH and administered in a suitable adjuvant such as Freunds. Preferably, selected peptides are coupled to a lysine core carrier substantially according to the methods of Tarn (1988) Proc Natl Acad Sci USA 85, 5409-5413. The resulting antibodies may be modified to a monovalent form e.g. Fab, FAB', or FV. Anti-idiotypic antibodies, especially internal imaging anti-idiotypic antibodies, may also be prepared using known methods. In one embodiment, purified TAF-145 is used to immunize mice, after which their spleens are removed, and splenocytes used to form cell hybrids with myeloma cells and obtain clones of antibody-secreted cells according to techniques that are standard in the art. The resulting monoclonal antibodies are screened using in vitro assays such as those described above for the following activities: binding to TAF- 145, inhibiting the incorporation of TAF- 145 into multimeric TAF complexes, and inhibiting the interaction between TAF- 145 and TBP. In another embodiment, the entire TAF complex is used as an immuogen as above, and the resulting monoclonal antibodies are screened for their activity in inhibiting the in vitro assembly of any component of the TAF complex. Anti-TAF antibodies may be used to identify and quantify TAF components, using immunoassays such as ELISA, EMIT, CEDIA, SLLFA, and the like. Anti-TAF antibodies may also be used to block the transcriptional function of TAF by inhibiting formation of complexes between TAF subunits or between assembled TAF complexes and other transcription components, or by immunodepleting cell extracts or transcription reactions of TAF components. In addition, these antibodies can be used to identify, isolate, and purify TAFs from different sources, and to perform subcellular and histochemical localization studies. High-Throughput Drug Screening The present invention encompasses the identification of agents useful in modulating fungal gene transcription, particularly the transcription of genes by RNA Polymerase H in a TAF-dependent manner. In a preferred embodiment, a high-throughput screening protocol is used to survey a large number of test compounds for their ability to interfere with TAF-dependent processes. Test inhibitory compounds are screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NT), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical library is available from Aldrich (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily produceable. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means. Useful inhibitory agents are identified with a range of assays employing TAF or TAF-encoding nucleic acids. As examples, protein binding assays, nucleic acid binding assays and gel shift assays are useful approaches. Preferably, TAF complexes or subunits as provided by the present invention may be used in in vitro binding assays with either TBP alone or with a combination or subcombination of TBP and general transcription factors (GTFs). For example, TAF complexes or subunit may be immobilized on microtiter dishes using methods that are standard in the art. The plates are then exposed to radiolabelled TBP e.g. 32 P-TBP in the absence or presence of candidate compounds. Conversely, TBP may be immobilized, and incubated with radiolabelled TAF in the absence or presence of candidate compounds. Oligonucleotides comprising TBP target sequences may be used in conjunction with TBP and TAF (see Example 2 below.) Postive "hit" compounds are those that inhibit TAF-TBP interaction. In these case, incu.-ition, washing, and radioactivity detection steps can be automated, allowing the scre :; ng of a large number of compounds, preferably at least about 1000 compounds per week. Once a particular test compound has been identified as described above, its activity is then confirmed by adding it to an in vitro transcription reaction, and measuring its effect on TAF-mediated activated transcription (see e.g. Example 1 below.) It is also contemplated that a useful agent may interfere with the function of TAF but not with TAF-TBP complex assembly. To screen for such compounds, other functional assays are used e.g. in vitro transcription reactions alone. Finally, a test compound identified as described above is tested for two properties: 1) Its ability to inhibit fungal growth; and 2) Its lack of effect on mammalian transcription. Fungal growth is measured by any method well-known in the art e.g. optical density of a liquid culture, or colony formation on agar. The lack of effect of a test compound on mammalian TAF-TBP interaction is tested by replacing yeast components with an analogous human in vitro transcription system as in e.g. Manley et al. (1980) Proc.Natl.Acad. Sci. USA 77:3855-3859. It will be understood that a compound that interferes with any aspect of TAF assembly or function is a likely candidate for an antifungal drug. Thus, in a manner similar to that described above for TBP-TAF-145 binding paradigm, binding assays can be routinely devised that measure the interaction of two or more TAF subunits with each other, or the interaction of one or more TAF subumts with other necessary transcription factors. According to the present invention, useful agents may be found within numerous chemical classes, though typically they are organic compounds, and preferably small organic compounds. Small organic compounds have a molecular weight of more than 50 yet less than about 2,500 daltons, preferably less than about 750, more preferably less than about 250 daltons. Exemplary classes include peptides, saccharides, steroids, and the like. The compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents. For example, where peptide agents are identified, they may be modified in a variety of ways to enhance their stability, such as using an unnatural amino acid, such as a D-amino acid, particularly D-alanine, by functionalizing the amino or carboxyl terminus, e.g., for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like. Other methods of stabilization may include encapsulation, for example, in liposomes, etc. Therapeutic Applications For therapeutic uses e.g. the treatment of fungal infections in mammals, the compositions and agents disclosed herein may be administered by any convenient way, e.g. parenterally, conveniently in a physiologically acceptable carrier, e.g., phosphate buffered saline, saline, deionized water, or the like. Typically, the compositions are added to a retained physiological fluid such as blood or synovial fluid. Alternatively, the compositions may comprise creams, ointments, lotions, or sprays for topical use. Generally, the amount administered will be empirically determined, typically in the range of about 10 to 1000 pg/kg of the recipient. For peptide agents, the concentration of will generally be in the range of about 100 to 500 ug/ml in the dose administered. Other additives may be included, such as stabilizers, bactericides, etc. These additives will be present in conventional amounts. Example 1: Identification of Yeast TAFs The studies described below were performed to identify a _ isolate transcriptional activator factors (TAFs) from S. cerevisiae. EXPERIMENTAL PROCED URES Seletive Depletion of an Essential Coactivator on a GST-yTBP Column A whole cell extract (WCE) from S. cerevisiae strain Y57 was prepared as described (Woontner, M. , et al. Mol. Cell. Biol. 11, 4555-4560 (1991)) except that the ammonium sulfate pellet was dissolved and dialyzed into Buffer T containing 0.1M potassium acetate. Buffer T is 20mM HEPES-KOH ph 7.6, 10 mM magnesium acetate, 5mM EGTA, 5 mM DTT and 20% (v/v) glycerol plus protease inhibitors: 0.5 ug/ml each of leupeptin. pepstatin A, aprotinin, antipain-HCl, chymostatin, bestatin; 2mM benzamidine-HCl and 0.5 mM PMSF. The WCE was diluted and chromatographed on a 2.5 ml column containing yeast TBP bound to glutathione S-tr. ^ferase (yTBP-GST) or, as a control, glutathione S- transferase alone (GST). The yeast T-yTBP plasmid was constructed by cloning the BamHI fragment from pASYHD (SchiK.dt, M. et al. Proc. Natl. Acad. Sci. U.S.A. 86, 7785-7789 (1989)) into pGEX-1. GS -yTBP and GST columns were prepared as described (Lin, Y-S. & Green, M.R. : ell 64, 971-981 (1991)). The WCE was diluted in hiffer T (0.1 M KOAc) and passed four times over the columns followed by washing with 5 volumes of 0.2 M KOAc buffer T. Bound prott ns were eluted with buffer T containing 2M kCl for analytical runs. Eluates and flowthrough fractions were dialyzed into buffer T (0.1M KOAc). Flowthrough fractions (4 ml), peak 2M KCl eluates (1-2 ml), and an aliquot of unfractionated WCE were collected and dialyzed in parallel. The transcriptional activating activity of each fraction was measured. Reaction ures conta * ' 8 ul of WCE olumn flowthrough r ictions and 5 ul of eluate fr s and were perform -d in the 3nce (+) or the at • (-) of 100 ng of GAL4-V ml of gel. Transcr n re s were performed, a imples processed as descn (Woontner, M. , et Mo; . Biol. 11, 4555-45 1991)). Briefly, reaction i .xtures (30 ul) containeu δ0-12« WCE or GST-yTBP n wt icut*'! :raction in 25 mM HEPES-KOH, pH 7.6, 10 rr . agnesium acetate, 2.5 mM ^ 2 mM DTT, 8-10% (v/v) glycerol, 90-110 mM potassium acetate, 5 mM phosr or -..one, 1U creatine kinase (Sigma), 10U RNasin, 100 ng of template, approximaieiy 100 ng of GAL4-VP16 fusion, protein where indicated. GAL4-VP16 was prepared as described (Chasman, D. I. et al. Mol. Cell. Biol. 9, 4746-4749 (1989)), except that acetate buffers were used and the protein was further purified by heparin-agarose chromatography. - P urification of the Coactivator Approximately 2 g of WCE protein was passed four to five times over a 40 ml GST-TBP column equilibrated in buffer T (0.1M KOAc), and the column was washed with 5 column volumes of T (0.2M KOAc). The column was eluted by step gradients of: 40 ml 0.5M KOAc, 40 ml 1.0 M KOAc, and 50 ml 2M KCL containing 0.003 % NP-40, all in Buffer T. Most of the activity eluted in the 2M KCL fraction. The fractions were dialyzed in buffer T (0.1 M KOAc.) containing 0.003 % NP-40. Aliquots were removed and analyzed by SDS-PAGE and visualized by Silver staining. Fractions were assayed for transcriptional activity by addition to a GST-yTBP flowthrough in the presence and absence of GAL4-VP16. The peak activity from six columns (Total WCE Protein load 12 g, 800 ml) were pooled (9.5 mg, 180 ml) and passed over a 3 ml PI 1 column equilibrated in T (0.1M KOAc). The flowthrough fraction (8.6 mg, 185 ml) was loaded onto a 5 ml Econopak S-cartridge (BioRAD) equilibrated in Buffer A (0.1). Buffer A is 20 mM HEPES-KOH pH 7.6, 2 mM DTT, 1 mM EDTA, 0.003 % (v/v) NP-40, 20% (v/v) glycerol and the molarity of potassium acetate indicated in parentheses. The S- column was washed and eluted with a linear gradient of A(0.1) to A(0.5). Peak fractions eluted as a broad peak from 0.2M to 0.35M KOAc, and were identified by transcription assays. The peak S-column fractions (2.1 mg, 36 ml) were dialyzed in buffer A(0.1) and loaded onto a 1 ml Econopak hydroxylapatite column equilibrated in buffer C (0.01M potassium phosphate pH 7.7, 0.2 mM EDTA, 2 mM DTT, 0.01 % (v/v) NP-40, 20% (v/v) glycerol), washed, and eluted with a linear gradient to 0.3M potassium phosphate. Peak fractions eluting between 0.15 M and 0.30 M were pooled and dialyzed into buffer A (0.1). Approximately 1 mg (14 mis) of protein was recovered following hydroxylapatite chromatography. To measure transcription activity of the final Hydroxylapatite fraction, the GST-yTBP flowthrough (75 ug of protein) was supplemented with increasing quantities of the hydroxylapatite fraction (approximately 50-500ng) in the presence (+) or absence (-) of 100 ng GAL4-VP16. In some cases, the hydroxylapatite fraction (150 uL) was analyzed on a Superose 6 gel filtration column (HR 10/30) in Buffer A (0.3M KOAc). Co-immunoprecipitation of Coactivator and TBP A yeast WCE was diluted and adjusted to 0.35 M KOAc with buffer T containing 1.0 M KOAc, and NP-40 was added to 0.1 % (v/v). To pre-clear the extract, 3 ml aliquots (40 mg/ml protein) were incubated for 6 hr with 0.1 ml of washed Protein A- agarose beads. Two micrograms of affinity purified pre-immune or α-TBP antibody (Poon, D. & Weil, P. A. J. Biol. Chem. 268, 15325-15328 (1993)) were added and incubated on ice for 5 hr. Ten microliters Protein A-agarose were added, and the incubation continued for 16 hr with rotation at 40 C. Prior to recovering the immune- complexes by centrifugation, 25 uL of Protein A beads were added as carrier. Beads were then washed four times with 1 ml of buffer T (0.9 M KOAc) containing 0.1 % NP- 40 (v/v). yTAFHs were eluted by washes of buffer T (2M KCl) plus 0.1 % NP-40 (v/v) and 0.1 mg/ml insulin, followed by dialysis into: 20 mM HEPES pH 7.5, 5 mM EGTA, 0.15M KOAc, 10 mM magnesium acetate, 1 mM DTT, 10% glycerol (v/v). Far Western Blotting The hydroxylapatite fraction was separated by SDS-PAGE and transferred to Immobilon-P membranes, and blocked in buffer T (0.2 M KOAc) containing 0.01 % 35 (v/v) NP-40 and 4% BSA for 3 hr at room temperature followed by 2 hr at 40°C. S- labeled yTBP and hTBP and its mutant derivatives (Ha, I. et al. Genes & Dev. 7, 1021- 1032 (1993)) were synthesized in rabbit recticulocyte lysate and wheat germ extract, respectively, and were diluted in incubation buffer (buffer T (0.2) plus 0.01 % (v/v) NP-40 and 0.5 % BSA). Blots were incubated with comparable amounts of labeled proteins estimated by fluorography of a SDS/PAGE gel. The membrane was incubated with probe at 40 C for 14-18 min, twice inincubation buffer without BSA for 15 min, and rinsed in buffer T (0.2) lacking glycerol for 20 sec. The membrance was air dried, and exposed directly to film for 8-24 hr. RNA Polymerase HI Transcription RNA polymerase HI activity was monitored by run off transcription of the met tRNA gene (Santos, T. & Zasloff, M. Cell 23, 699-709 (1981)), and were performed similar to the RNA polymerase H transcription reactions using 300 ng of template except that RNAse Tl treatment was omitted and guanosine was included in the rNTP mixes. A yeast WCE and the GST-yTBP flowthrough were tested for their ability to support transcription of the tRNA gene. The GST-yTBP flowthrough was also supplemented with the GST-yTBP 2M KCl eluate (5 uL) or the hydroxylapatite fraction (3 uL) as indicated. Cloning genes encoding TAF proteins The hydroxylapatite fraction of yTAFHs was subjected to SDS/PAGE, the polypeptides transferred to nitrocellulose, and the bands excised for microsequence analysis. A peptide with the sequence ATTEPSAEPDEPFIGYLGDVTA SEQ. I.D. No. 5 was obtained from yTAFH90. A BLAST search (Altschul, S.F. et al. J. Mol. Biol. 215, 403-410 (1990)) of the database identified yTAFH90 as an unknown open reading frame on yeast chromosome HI. yTAFH90 was cloned by PCR from genomic DNA using the primers: 5 -AGATACTTGAAAATCTAGAATGTCACAC-3' SEQ. I.D. No. 6 and 5'-GGTTATACTTATTACATATCACTTCCATGC-3'SEQ. I.D. No. 7. The peptides MNHI_FTVGQTFPVEEIPGPN SEQ. I.D. No. 8 and LPVGETHVLGVQDKSPF SEQ. I.D. No. 9 were obtained from yTAFH145 and the degenerate oligonucleotides 5'-TT(C/T)CCNGTNGA(A/G)GA(A/G)AT-3' SEQ. I.D. No. 10 and 5'- GTNGGNGA(A/G)ACNCA(C/T)GT-3' SEQ. I.D. No. 11 were designed and used to screen a yeast genomic library (Rose, M.D. et al. Gene 60, 237-243). Two clones were selected that contained the same 10 kb insert. The open reading frame was identified by sequencing and by its homology to drosophila and human TAFII250. No other extended ORFs were found in the sequence. Genetic Analysis A disrupted copy of the yTAFH90 gene was generated by insertion of the HIS 3 gene (Bam HI fragment) into the Bgl H site (amino acid 489) and Bam HI site (amino acid 628) within the coding region, and a disrupted copy of the yTAFH145 gene was constructed by insertion of the LEU2 gene (blunted Bam HI/Hind HI fragment) into the blunted Bgl π/Cla I sites (amino acid 233 and 645). These fragments were used to transform the strain CY236 to histidine (strain LY1) or leucine (strain YSW57) prototrophy. RESULTS Identification of a Yeast Coactivator that Binds TBP To identify yeast coactivators that bind TBP, protein affinity- chromatography was carried out using S. cerevisiae TBP (yTBP) as the immobilized ligand. An S. cerevisiae whole cell extract (yWCE) was chromatographed on a control glutathione S-transferase (GST) or a GST-yTBP column. The flowthrough fraction was tested for its ability to support transcription in the presence or absence of the activator GAL4-VP16. The DNA template used in these experiments contained the yeast cycl promoter and a single GAL4 binding site upstream of the TATA box (Figure 1, bottom; Kelleher, R. J., Flanagan, P. M. & Kornberg, R. D. Cell 61, 1209-1215 (1990)). Figure 1 shows that the flowthrough of the GST column supported both basal (lane 3) and activated (lane 4) transcription. In co st, the flowthrough of a GST- yTBP column did not support activated transcription (Ian. 0), whereas basal transcription remained intact (lane 9). Thus, under these chromatographic conditions, yTBP select 5 ely depleted a coactivator from the yWCE. Addition of the 2M KCl eluate from the GST-yTBP column restored the ility of the GST-TBP flowthrough to respond to GALΛ-VP16 (lane 14), whereas the eluate of the GST column had no effect (Figure 1 and data not shown). The 2M KCl eluate from the GST-yTBP column had no transcriptional activity on its own (lanes 11 and 12), and did not significantly affect activator-independentbasal transcription (lane 13). Taken together, these data identify an essential yeast coactivator activity that binds TBP. Purification of the Coactivator The coactivator implicated by the protein affinity-chromatography experiments was purified next. The GST-yTBP column was eluted with salt, and the fractions tested for their ability to restore activated transcription to the GST-yTBP flowthrough. The majority of bound protein eluted from the yTBP affinity column at 0.5M potassium acetate, whereasmost of the coactivator activity eluted in the 2M KCl fraction, which contained only 0.07% of the total protein (data not shown). The coactivator was further purified from the GST-yTBP 2M KCl eluate accordi co the scheme outlined in Figure 2A. The fractions with maximal coactivator activity from each column were identified by a transcription assay performed in the presence or absence of GAL4-VP16. The polypeptide composition of these peak fractions from each column is shown in Figure 2B. The final franction from the hydroxylapatite column contained approximately eleven major polypeptides, ranging from 30-200kD, and a number of minor peptides. Nine of these polyeptides, (180kD, 145kD, 116kD, 90kD, 68kD, 62kD, 51-54kD, 47kD, 47kD and 30kD) cofractionated with the transcriptional activity (Figure 2B and data not shown), and co-eluted on a Superose 6 gel filtration column (see below). Figure 2C shows that addition of the final hydroxylapatite fraction to the GST-yTBP flowthrough fraction restored, in a dose-dependent fashion, the ability of GAL4-VP16 to activate transcription (lanes 6-8). In contrast, equivalent amounts of this fraction had not effect on basal transcription activity (lanes 3-5). The co-elution of these polypeptides over the four chromatographic columns in Figure 2 suggested that they may be components of a single complex. To confirm this possibility, the final hydroxylapatite fraction was analyzed by gel filtration chromatography. Figure 3 A shows that on a Superose 6 gel filtration column nine of the polypeptides eluted as a complex with a native molecular mass of 800kD (fraction 14). Two of the eleven major polypeptides contained in the hydroxylapatite fraction, 200kD and 50kD, eluted separately in fractions 20 and 22-24, respectively. These two polypeptides also eluted slightly ahead of the complex on the hydroxylapaptite column (data not shown). Figure 3 A also reveals a cluster of polypeptides of 51-54kD that eluted with the complex but were not well visualized in the hydroxylapatite fraction due, at least in part, to masking by the 50kD contaminant (Figure 2B). Significantly, this 51-54kD cluster of polypeptides was also co-immunoprecipitated with an α-TBP antibody (see below) and they are believed to be components of the complex. These combined data indicated that the coactivator is contained within a complex of approximately nine polypeptides, which we refer to as the S. cerevisiae TAF (yTAF) complex. yTAFs Co-immuniprecipitate with TBP To provide additional evidence that the coactivator bound TBP, co- immunoprecipitation experiments were carried out using an α-TBP antibody. Immuniprecipitations were carried out in a yeast whole cell extract and the 8immuniprecipitates analyzed for their polypeptide composition by SDS-PAGE and silver staining (Figure 3B), and for their ability to restore activated transcription to the GST- yTBP flowthrough (Figure 3C). Strikingly, eight of the nine major polypeptides present in the chromatographically purified yTAF complex were clearly co-immunoprecipitated by the or-TBP antibody and not by the control pre-immune serum (Figure 3B). The sole exception was yTAFllό, which could result from dissociation during washing of the immune-complexes . Significantly , the immunoprecipitate contained the cluster of 51 -54kD polypeptides discussed above. The following experiment was carried out to determine whether the immuniprecipitate contained the coactivator activity. Immune-complexes immobilized on protein A agarose beads were washed with 2M KCl, which released the TBP-associated polypeptides but not TBP. Figure 3C shows that the 2M KCl wash of the immune-compl-xes partially restored activitated transcription to the GST-yTBP flowthrough (lanes 11-12) without affecting the basal level (lanes 7-8). Thus, an activity required for activated transcription was co-immunoprecipitated with TBP. The reduced coactivator activity of the immune-complex eluate, compared to the hydroxylapatite fraction (compare Figures 2C and 3C), is consistent with the 5-8 fold lower concentration of yTAFs in the immune-complex eluate (see Figure 3B and legend). The C-Terminal Domain of TBP Binds to yTAF145 The results from the protein affinity-chromatography and co- immunoprecipitation experiments described above strongly suggested that one or more subumts of the yTAF complex bound TBP directly. To identify this subunit, the yTAF complex was examined for TBP binding using an immobilized protein blotting ("Far Western") assay. The hydroxylapri's fraction was fractionated by SDS-PAGE, the 35 polypeptides transferred to nitrocellulose, and the membrane probed with S-labeled yTBP. Figure 4 shows that a single subunit of the yTAF complex, the 145kD polypeptide 35 (yTAF145), was bound by yTBP. Figure 4 sl ows that S-labeled human TBP also bound to yTAF 145. The fact that both human and S. cerevisiae TBP bind to yTAF 145 implies that the interaction occurs through the C-terminal domain of TBP, which is conserved between human and S. cerevisiae TBP. To map the region of TBP required for binding to yTAF145, we analyzed several previously characterized human TBP mutants (Ha, I. et al. Genes & Dev. 7, 1021-1032 (1993)). Deletion of most of the second direct repeat led to a significant reduction in the interaction between TBP and yTAF 145 (Figure 4B; lane 2), and deletion of another 50 amino acids totally abolished binding (lane 3). Significantly, this same region was shown to bind hTAF250. A mutant lacking most of the second direct repeat, but containing the first direct repeat and the basic region of TBP also failed to bind yTAF145 (169-220; lane 6). Therefore, it appears that TBP interacts with yTAF145 through both direct repeats, consistent with the OsaddleO structure of TBP (Nikolov, D.B. et al. Nature 360 40-46 (1992); Chasman, D.I. et al. Proc. Natl. Acad. Sci. USA 90, 8174-8178 (1993)). yTAFs are Not Required for Transcription by RNA Polymerase III In higher eukaryotes, each of the three RNA polymerases contains an essential transcription factor composed of TBP and multiple TAFs. Furthermore, the TAFs involved in transcription by RNA polymerase H, TAFs, are distinct from those involved in transcription by the other two RNA polymerases (reviewed in Hernandez, N. Genes & Dev. 7, 1291-1308 (1993)). In yeast, TBP is also required for transcription by all three RNA polymerases (Cormack, B. P. & Stnihl, K. Cell 69, 685-696 (1992); Schultz, M. C, Reeder, R. H. & Hahn, S. Cell 69, 697-702 (1992). The experiments described above demonstrate that a GST-yTBP column depleted a S. cerevisiae WCE of TAFs with coactivator activity. The ability of the GST- yTBP flowthrough to support RNA polymerase IH-directed transcription was tested to determine whether these TAFs were required for transcription by RNA polymerase HI. Figure 5 shows that the GST-yTBP flowthrough efficiently supported RNA polymerase HI transcription, and that this activity was not increased by addition of either the GST- yTBP eluate, or the purified yTAF complex. These combined data demonstrate that the GST-yTBP column selectively depleted a factor required for transcriptional activation by RNA polymerase H. It is, of course, possible that under different chromatorgraphic conditions the GST-yTBP column could also deplete TAFs involved in RNA polymerase HI transcription. Yeast Homologues of Higher Eukaryotic TAFs The yTAF complex was purified as described above and the polypeptides isolated for microsequence analysis. A protein database search of a tryptic peptide derived from yTAF90 revealed that it was present within a previously sequenced gene on yeast chromosome HI (Oliver, S. G. et al. Nature 357, 38-46 (1992)). The amino acid sequence of yTAF90 contains WD40, or /3-transducin repeats (van der Voom, L. & Ploegh, H.L. FEBS Lett. 307, 131-134 (1992)). Drosophila TAF80 (dTAF80) also contains these motifs, and yTAF90 and dTAF80 are somewhat homologous (Figure 6A); yTAF90 has 31 % identity and 53 % similarity with dTAF80, a degree of homology equivalent no greater than that between human and yeast TFHB, which are analogous but not interchangeable (Pinto, I. , Ware, D. E. & Hampsey, M. Cell 68, 977-988 (1992)). The similar size and sequence indicates that yTAF90 is the yeast homologue of dTAF80. Two peptide sequences were obtained from yTAF 145, degenerate oligonucleotides were designed and used to screen a plasmid yeast genomic DNA library. DNA sequence analysis of positive clones revealed an open reading frame of 1066 amino acids, with a calculated molecular weight of 120,685 kDa. This value is somewhat smaller that the molecular weight estimated by polyacrylamide gel electrophoresis (145 kDa), possibly due to the highly charged nature of yTAF145. The N-terminus of yTAF145 is highly acidic (pi 3.3), while the C-terminus is rich in basic residues (pi 9.3). A data base search of the sequence revealed that it has some homology to both human (Figure 6B) and drosophila (data not shown) TAF250. Figure 6B (bottom) highlights regions of similarity and difference between yTAF145 and hTAF250. Both contain a highly acidic N-terminus rich in serines, homology to bacterial sigma factors, and homology to high mobility group proteins. The C-terminal portion of human and drosophila TAF250 contain "Bromo" domains and an acidic region, which have been proposed to provide a transcription related function (Hisatake, K. et al. Nature 362, 179-181 (1993); Ruppert, S., Wang, E. H. & Tjian, R. Nature (London) 362, 175-179 (1993); Kokubo, T., et al. Genes & Dev. 7, 1033-1046 (1993)). Significantly, this C-Terminal region is absent in yTAF145, indicating an important difference between the yeast and human TAFs. The Genes Encoding yTAF90 and yTAF145 are Essential The following procedure was performed to determine whether the genes encoding yTAF90 or yTAF145 are required for cell viability by genetic analysis of strains bearing a disrupted allele of these genes. Constructs containing disrupted copies of yTAF90 and yTAF145 were assembled and used to make diploid yeast strains containing one wildtype and one mutant copy of either gene (Figure 6, panels C And D, top). The disruptions were confirmed by Southern blotting (Figure 6, panels C and D, left). These strains were sporulated and their tetrads dissected. The auxotrophic markers for each segregated 2:2 (Figure 6, panels C and D, bottom right), and in no case did a spore bearing the disruption marker grow, indicating that these genes are essential for cell viability. DISCUSSION Identification of fungal coactivators is important for establishing bioassays for the discovery of novel anti-fungal therapeutics. To date, the most well characterized coactivators are the TAFs of the higher eukaryotic TFHD complex (reviewed in Pugh, B. F. & Tjian, R., J. Biol. Chem. , 267:679-682 (1992); Sharp, P. A., Cell, 68:819-821 (1992); Hernandez, N., Genes & Dev. , 7:1291-1308 (1993)). A novel yeast complex has been identified that shares several similarities with higher eukaryotic TFHD, including: (1) both are large multisubunit complexes that contain a single subunit that binds TBP directly; (4) both complexes appear to be specific for RNA polymerase H transcripton; (5) two subunits of the yeast complex, yTAF90 and yTAF145, are the apparent homologues of known subunits of higher eukaryotic TFHD. Differences between TFHD isolated from higher eukaryotes and the yTAF complex described here are exemplified best by the fact that TBP is an integral component of TFHD but is absent from the purified yTAF complex. This apparent disparity is believed ato reflect only a difference in the affinity of TBP for the two complexes (Figure 6E). In higher eukaryotic TFHD, TBP interacts with hTAF25023-27 whereas in the yTAF complex it is the homologous yTAF145 with which TBP interacts (Figure 4). Presumably, the relatively weak affinity of TBP for yTAF145 has made it previously difficult to identify yeast TAFs. The weak interaction between yTBP and the yTAF complex demonstrates the possibility for therpeutic intervention. For example, some natural protein negative regulators of transcription interact directly with TBP to prevent either DNA binding (Auble, D.T. & Hahn, S., Genes & Dev. , 7:844-856 (1993)), or the interaction of TBP with other GTFs Inostraoza, J. A.et al., Cell, 70:477-489 (1992); Meisteremst, M. & Roeder, R. G. , Cell, 67:557-567 (1991); Meisteremst, M. et al., Cell, 66:981-993 (1991)). By analogy, it is conceivable that an antifungal drug could be developed that binds to free yTAF and blocks its interaction with yTBP or that binds to yTBP and blocks its interaction with the yTAF complex. The relatively weak interaction between TBP and the yTAF complex may facilitate disruption of TAF function. Other Yeast TBP-Binding Proteins It is important that the yTAF complex disclosed here is distinct from previously identified yeast transcription factors. Genetic and biochemical approaches have identifed four factors that interact with yeast TBP (Pinto, I. , Ware, D. E. & Hampsey, M., Cell, 68:977-988 (1992); Colbert, T. & Hahn, S., Genes & Dev. , 6: 1940-1949 (1992); Eisenmann, D. M.et al., Genes & Dev. , 61319-1331 (1992); Lopez-de-Leon et al. , Cell, 71:211-220; Kassavetis, G. A. et al. , Cell, 71: 1055-1064 (1992); Koleske, A. J. et al. , Cell, 69:883-894 (1992); Poon, D. & WeU, P. A., J. Biol. Chem. , 268:15325- 15328 (1993)). Two of these factors, the essential RNA polymerase H transcription factor TFHB (SUA7), and the essential RNA polymerase HI transcription factor BRF1/PCF4/TDS4, are unrelated to the yTAF complex based upon transcriptional assays (Figures 1 , 2C and 5) and microsequence analysis (data not shown The yc protein, SPT3 interacts with TBP on tϊh .isis of genetic and biochemical experim H 'ever, unlike the coactivator activity reported here, SPT3 is not selectively requ_.ed for activated transcription (Eisenmann, D. M.et al., Genes & Dev. , 6: 1319-1331 (1992)). Furthermore, immunoblotting experiments indicate that the purified yTAF complex does not contain SPT3. Recently, a yeast multisubunit complex has been identified that contains RNA polymerase H and a minimum of twelve other subumts (Thompson, C. M. et al. , Cell, 73: 1361-1375 (1993); Kim, Y. -J., et al. , Cell, 77:599-608 (1994)). This holoenzyme complex contains a group of polypeptides (SRB2, 4, 5 and 6) that had been previously identified as dominant mutations that suppress certain RNA polymerase H C- terminal domain deletion mutants. One of these proteins, SRB2, appears to interact with TBP (Koleske, A. J. et al., Cell, 69:883-894 (1992)). Unlike the yTAFs disclosed here, the SRB proteins are necessary for both basal and activated transcription (Koleske, A. J. et al. , Cell, 69:883-894 (1992); Thompson, C. M. et al. , Cell, 73: 1361-1375 (1993)). Furthermore, immunoblotting experiments indicate that SRB2, SRB4, SRB5, and SRB6 are not present in the purified yTAF complex. A recent study (Verrijzer, C.P. et al. Science 264, 933-94 (1994)) reported that a cloned Drosophila TAF, dTAFι50, bears significant homology to the product of the yeast TSM-1 gene. This study also demonstrated that the TSM-1 protein can bind purified TBP(Veπijzer, C.P. et al., Science, 264:933-941 (1994)). However, microsequence analysis indicates that TSM-1 and the yTAF complex disclosed here are distinct entities. Furthermore, we find that TBP binds to only one subunit of the yTAF complex, yTAF145. Consistent with our results in yeast, immunopurified human TFHD appears to lack TAF150 (Hisatake, K. et al. , Nature, 362:179-181 (1993); Zhou, Q. , Boyer, T. G. & Berk, A. J., Genes & Dev. , 7: 180-187 (1993)) and the presence of TAF150 in drosophila TFHD is variable (Kokubo, T. , et al. , Genes & Dev. , 7: 1033-1046 (1993)). Thus, whether or not TSM-1 is a TAF, and the role of TSM-1 in transcription, are issues that remain to be clarified. Other Yeast Coactivators Several laboratories have identifed yeast coactivator activities (Ha, I. et al. , Genes & Dev. , 7: 1021-1032 (1993); Koleske, A. J. & Young, R. A. , Nature, 368:466- 469 (1994); S. L.et al., Cell, 70:251-265 (1992)) which could be the same or different from yTAFs: some human coactivators, such as USA (Meisteremst, M. & Roeder, R. G., Cell, 67:557-567 (1991); Meisteremst, M. et al., Cell, 66:981-993 (1991) and ACF (Merino, A. et al. , Nature, 3j}5:227-232 (1993)), are clearly distinct from the known TAFs. The RNA polymerase holoenzyme complex described above was recently shown to direct activator mediated transcription when supplemented with purified yeast GTFs (Kim, Y. -J. , et al. , Cell, 77:599-608 (1994)), and recent studies have shown that this holoenzyme is indistinquishable from a previously biochemically defined Omediator fractionό (Kim, Y. -J., et al., Cell, 77:599-608 (1994)). Genetic studies have led to the identification of a putative coactivator, ADA242. However, yTAF90 and yTAF 145 are essential, wheras ADA2 is not. Furthermore, there is no evidence that ADA2 interats with TBP. Yeast Contains Multiple TAF Complexes TBP is a component of the yeast RNA polymerase HI transcription factor, TFIHB (reviewed in Hernandez, N., Genes & Dev. , 7:1291-1308 (1993)). Recently, Poon, D. & Weil, P. A. , J. Biol. Chem. , 268: 15325-15328 (1993) have identified a yeast TBP-containing complex required for transcription by RNA polymerase HI. This complex contains eight major subumts, including a 70kD polypeptide that is BRF1/PCR4/TDS4, a known component of TFIHB. These observations strongly suggest that the complex identified by Poon and Weil is, or is closely related to, TFIHB. While it is possible that there are polypeptides in common, several observations indicate that the yTAF complex identified in this study is distinct from the complex described by Poon and Weil. First, the GST-yTBP column failed to deplete an extract of its ability to support transcription by RNA polymerase HI, and, conversely, addition of the purified yTAF complex to the GST-yTBP flowthrough did not enhance transcription by RNA polymerase HI (Figure 5). Second, microsequence analysis of yTAF68 confirms that it is not BRF1/PCF4/TDS4 (our unpublished data). Thus, these combined data indicate that yeast contain at least two TBP-binding complexes, required for transcription by RNA polymerase HI, and RNA polymerase H (disclosed here). The identification and cloning of yTAFs provides the opportunity to develop novel antifungal agents using biochemical and genetic screening approaches. For example, the discovery that yTAF90 and yTAF 145 are essential for growth and viability of yeast is of particular interest because it proves that drugs blocking the function of either of these proteins will block the growth of fungi. It provides the necessary tools to establish in vitro and cell-based screens for therapeutics. Example 2: High-throughput Screening of Anti-TAF compounds Corning ELISA strip wells (8 wells per strip) are coated with avidin (1.0 ug per well) by incubating avidin (200 ul of a 5 ug/ml stock) in coupling buffer (per liter: 1.6g Na 2 CO 3 , 2.9 g , NaHCO 3 , 0.9 g NaN 3 ) on the well for 12 h at 4°C. The buffer is decanted, and nonspecific binding sites on the wells are blocked with 1 % skim milk in phosphate-buffered saline (PBS) for 1 h at 37 °C. Blocking buffer is discarded, and a yeast Pol H promoter-containing oligonucleotide (1 pmol/well) is added to the wells and incubated for 30 min at room temperature. The oligonucleotide is double-stranded and contains a biotin tag on the sense strand. The otøgonucleotide-containing solution is then removed, and the wells are washed with 1 % milk in PBS. Yeast TBP is mixed with partially purified TAF that had been metabolically labelled with 35 S-methionine or purified TAF-145 similarly labelled, all in HEG buffer (0.1 M KCl, 25 mM HEPES pH 7.9, 0.5 mM EDTA, 20% glycerol, 0.01 % LDAO, 0.1 M AEBSF, 0.1 M Na metabisulfite, 10 mM |3-mercaptoethanol) plus 200 ug/ml bovine serum albumin (BSA). The protein mixture is then added to the prepared wells and incubated for 30 min at room temperature. Samples are then removed, and the wells are washed three times with the PBS/milk solution. Wells are separated and put into scintillation vials, scintillation cocktail is added, and samples are counted in a liquid scintillation counter. Binding of yeast TAF to the wells is found to be dependent on the presence of TBP, bound in turn to the Pol H promoter-containing oligonucleotide. Small molecules, whether purified or present in natural or synthetic mixtures, are introduced into the assay at concentrations ranging from about 20 to about 200 μM, and appropriate solvent controls are also performed. Compounds that inhibit binding of TAF by more than about 30% are identified, and the inhibitory activity purified if not already available in pure form. Compounds identified as described above are then tested for their ability to inhibit TBP-dependent transcription in a mammalian cell-free system. Alternatively, human TBP is used in place of yeast TBP in the above-described assay for TAF binding to TBP. Thus, a subset of active compounds are identified that selectively interfere with yeast TAF function i.e. are fungal-specific. Example 3: Treatment of Fungal Infections A compound identified by the methods described in Example 2 as possessing anti-yeast TAF activity is titrated for its fungal growth-inhibiting properties. That is, the concentration range in which the compound effectively suppresses the growth of Candida albicans is measured using methods that are standard in the art. The toxicity of the compound for mammalian cells over the identical concentration range is then tested using standard procedures. A pharmaceutical formulation is prepared in sterile saline containing the above compound in a concentration effective to prevent growth of Candida organisms without affecting the viability or function of mammalian cells i.e. at a concentration at which the compound exhibits minimal or no toxicity, or toxicity at a level generally accepted in the art. The above formulation is administered using an intravenous, intramuscular, or subcutaneous route to treat candidiasis in a mammalian animal. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: UNIVERSITY OF MASSCHUSETTS MEDICAL CENTER (ii) TITLE OF INVENTION: A Novel Fungal Multisubunit Protein Complex Critical for Expression of Fungal Proteins (iii) NUMBER OF SEQUENCES: 11 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Darby & Darby PC (B) STREET: 805 Third Avenue (C) CITY: New York (D) STATE: New York (E) COUNTRY: US (F) ZIP: 10022 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US 08/308.818 (B) FILING DATE: 19-SEP-1994 SUBSTITUTE SHEET (RULE 26Ϊ (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Ludwig, S. Peter (B) REGISTRATION NUMBER: 25.351 (C) REFERENCE/DOCKET NUMBER: 0342/0A404 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 212-52707700 (B) TELEFAX: 212-753-6237 (C) TELEX: 236687 (2) INFORMATION FOR SEQ ID NO: l : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1066 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: S. cerevisiae (vii) IMMEDIATE SOURCE: (B) CLONE: TAF- 145 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l : Met Val Lys Gin Gin Gly Ser Gly Lys Thr Asn Leu Ala Asn Glu Asp 1 5 10 15 Glu Ala Tyr Glu Ala He Phe Gly Gly Glu Phe Gly Ser Leu Glu He 20 25 30 Gly Ser Tyr He Gly Gly Asp Glu Gly Ala Asn Ser Lys Asp Tyr Thr 35 40 45 Glu His Leu Pro Asp Ala Val Asp Phe Glu Asp Glu Asp Glu Leu Ala 50 55 60 Asp Asp Asp Asp Asp Leu Pro Glu Glu Ser Asp Ala Asn Leu His Pro 65 70 75 80 Ala Met Met Thr Met Gly Ala Tyr Asp Asp Val Asn Glu Asn Gly Ala 85 90 95 Val Leu Gly He Asp Ser Asn Ser Leu Asn Met Gin Leu Pro Glu He 100 105 110 Asn Gly Asp Leu Ser Gin Gin Phe He Leu Glu Asp Asp Gly Gly Thr 115 120 125 Pro Ala Thr Ser Asn Ala Leu Phe Met Gly Met Asp Ala Asn Glu He 130 135 140 His Leu Ala Thr Glu Thr Gly Val Leu Asp Gly Ser Gly Ala Asn Glu 145 150 155 160 He Gly His Ser Gin Leu Ser He Gly Gly Val Asn Gly Asn Asp Met 165 170 175 Ser He Asn Gly Gly Phe He Met Glu Pro Asp Met Ser Asp Gly Lys 180 185 190 His Lys Lys Ala Thr Lys Leu Asp Leu He Asn His Glu Lys Tyr Leu 195 200 205 Leu Lys Lys Tyr Phe Pro Asp Phe Glu Lys Gly Lys He Leu Lys Tφ 210 215 220 Asn Lys Leu He Tyr Arg Arg Ser Val Pro Tyr His Tφ His Ser Glu 225 230 235 240 He Ser Arg Val Lys Lys Pro Phe Met Pro Leu Asn Leu Lys Phe Lys 245 250 255 Val Gin Gin Asp Asp Lys Arg Leu Phe Asn Ser Arg Thr He Ser Tyr 260 265 270 Val Ala Pro He Tyr Gin Gly Lys Asn Asn Leu Leu Gin Ser Asn Ser 275 280 285 Ser Ala Ser Arg Arg Gly Leu He His Val Ser He Asp Glu Leu Phe 290 295 300 Pro He Lys Glu Gin Gin Lys Lys Arg Lys He He His Asp Glu Lys 305 310 315 320 Thr He Ser Glu Asp Leu Leu He Ala Thr Asp Asp Tφ Asp Gin Glu 325 330 335 Lys He He Asn Gin Gly Thr Ser Ser Thr Ala Thr Leu Ala Asp Ser 340 345 350 Ser Met Thr Pro Asn Leu Lys Phe Ser Gly Gly Tyr Lys Leu Lys Ser 355 360 365 Leu He Glu Asp Val Ala Glu Asp Tφ Gin Trp Asp Glu Asp Met He 370 375 380 He Asp Ala Lys Leu Lys Glu Ser Lys His Ala Glu Leu Asn Met Asn 385 390 395 400 Asp Glu Lys Leu Leu Leu Met He Glu Lys Thr Asn Asn Leu Ala Gin 405 410 415 Gin Lys Gin Gin Leu Asp Ser Ser Asn Leu He Leu Pro Leu Asn Glu 420 425 430 Thr He Leu Gin Gin Lys Phe Asn Leu Ser Asn Asp Asp Lys Tyr Gin 435 440 445 He Leu Lys Lys Thr His Gin Thr Lys Val Arg Ser Thr He Ser Asn 450 455 460 Leu Asn He Gin His Ser Gin Pro Ala He Asn Leu Gin Ser Pro Phe 465 470 475 480 Tyr Lys Val Ala Val Pro Arg Tyr Gin Leu Arg His Phe His Arg Glu 485 490 495 Asn Phe Gly Ser His He Arg Pro Gly Thr Lys He Val Phe Ser Lys 500 505 510 Leu Lys Ala Arg Lys Arg Lys Arg Asp Lys Gly Lys Asp Val Lys Glu 515 520 525 Ser Phe Ser Thr Ser Gin Asp Leu Thr He Gly Asp Thr Ala Pro Val 530 535 540 Tyr Leu Met Glu Tyr Ser Glu Gin Thr Pro Val Ala Leu Ser Lys Arg 545 550 555 560 Gly Met Ala Asn Lys Leu He Asn Tyr Tyr Arg Lys Ala Asn Glu Gin 565 570 575 Asp Thr Leu Arg Pro Lys Leu Pro Val Gly Glu Thr His Val Leu Gly 580 585 590 Val Gin Asp Lys Ser Pro Phe Tφ Asn Phe Gly Phe Val Glu Pro Gly 595 600 605 His He Val Pro Thr Leu Tyr Asn Asn Met He Arg Ala Pro Val Phe 610 615 620 Lys His Asp He Ser Gly Thr Asp Phe Leu Leu Thr Lys Ser Ser Gly 625 630 635 640 Phe Gly He Ser Asn Arg Phe Tyr Leu Arg Asn He Asn His Leu Phe 645 650 655 Thr Val Gly Gin Thr Phe Pro Val Glu Glu He Pro Gly Pro Asn Ser 660 665 670 Arg Lys Val Thr Ser Met Lys Ala Thr Arg Leu Lys Met He He Tyr 675 680 685 Arg He Leu Asn His Asn His Ser Lys Ala He Ser He Asp Pro He 690 695 700 Ala Lys His Phe Pro Asp Gin Asp Tyr Gly Gin Asn Arg Gin Lys Val 705 710 715 720 Lys Glu Phe Met Lys Tyr Gin Arg Asp Gly Pro Glu Lys Gly Leu Tφ 725 730 735 Arg Leu Lys Asp Asp Glu Lys Leu Leu Asp Asn Glu Ala Val Lys Ser 740 745 750 Leu He Thr Pro Glu Gin He Ser C Val Glu Ser Met Ser Gin Gly 755 760 765 Leu Gin Phe Gin Glu Asp Asn Glu Ala Tyr Asn Phe Asp Ser Lys Leu 770 775 780 Lys Ser Leu Glu Glu Asn Leu Leu Pro Tφ Asn He Thr Lys Asn Phe 785 790 795 800 He Asn Ser Thr Gin Met Arg Ala Met He Gin He His Gly Val Gly 805 810 815 Asp P fhr Gly Cys Gly Glu Gly Phe S.r Phe Leu Lys Thr Ser Met 820 825 830 Lys Gly Gly Phe Val Lys Ser Gly Ser Pro Ser Ser Asn Asn Asu Ser 835 840 845 Ser Asn Lys Lys Gly Thr Asn Thr His Ser Tyr Asn Val Ala Gin Gin 850 855 860 Gin Lys Ala Tyr Asp Glu Glu He Ala Lys Thr Tφ Tyr Thr His Thr 865 870 875 880 Lys Ser Leu Ser He Ser Asn Pro Phe Glu Glu Met Thr Asn Pro Asp 885 890 895 Glu He Asn Gin Thr Asn Lys His Val Lys Thr Asp Arg Asp Asp Lys 900 905 910 Lys He Leu Lys He Val Arg Lys Lys Arg Asp Glu Asn Gly He He 915 920 925 Gin Arg Gin Thr He Phe He Arg Asp Pro Arg Val He Gin Gly Tyr 930 935 940 He Lys He Lys Glu Gin Asp Lys Glu Asp Val Asn Lys Leu Leu Glu 945 950 955 960 Glu Asp Thr Ser Lys He Asn Asn Leu Glu Glu Leu Glu Lys Gin Lys 965 970 975 Lys Leu Leu Gin Leu Glu Leu Ala Asn Leu Glu Lys Ser Gin Gin Arg 980 985 990 Arg Ala Ala Arg Gin Asn Ser Lys Arg Asn Gly Gly Ala Thr Arg Thr 995 1000 1005 Glu Asn Ser Val Asp Asn Gly Ser Asp Leu Ala Gly Val Thr Asp Gly 1010 1015 1020 Lys Ala Ala Arg Asn Lys Gly Lys Asn Thr Thr Arg Arg Cys Ala Thr 1025 1030 1035 1040 Cys Gly Gin He Gly His He Arg Thr Asn Lys Ser Cys Pro Met Tyr 1045 1050 1055 Ser Ser Lys Asp Asn Pro Ala Ser Pro Lys 1060 1065 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 798 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: S. cerevisiae (vii) IMMEDIATE SOURCE: (B) CLONE: TAF-90 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ser Gin Lys Gin Ser Thr Asn Gin Asn Gin Asn Gly Thr His Gin 1 5 10 15 Pro Gin Pro Val Lys Asn Gin Arg Thr Asn Asn Ala Ala Gly Ala Asn 20 25 30 Ser Gly Gin Gin Pro Gin Gin Gin Ser Gin Gly Gin Ser Gin Gin Gin 35 40 45 Gly Arg Ser Asn Gly Pro Phe Ser Ala Ser Asp Leu Asn Arg He Val 50 55 60 Leu Glu Tyr Leu Asn Lys Lys Gly Tyr His Arg Thr Glu Ala Met Leu 65 70 75 80 Arg Ala Glu Ser Gly Arg Thr Leu Thr Pro Gin Asn Lys Gin Ser Pro 85 90 95 Ala Asn Thr Lys Thr Gly Lys Phe Pro Glu Gin Ser Ser He Pro Pro 100 105 110 Asn Pro Gly Lys Thr Ala Lys Pro He Ser Asn Pro Thr Asn Leu Ser 1 15 120 125 Ser Lys Arg Asp Ala Glu Gly Gly He Val Ser Ser Gly Arg Leu Glu 130 135 140 Gly Leu Asn Ala Pro Glu Asn Tyr He Arg Ala Tyr Ser Met Leu Lys 145 150 155 160 Asn Tφ Val Asp Ser Ser Leu Glu He Tyr Lys Pro Glu Leu Ser Tyr 165 170 175 He Met Tyr Pro He Phe He Tyr Leu Phe Leu Asn Leu Val Ala Lys 180 185 190 SUBSTITUTE SHEET (RULE 26 Asn Pro Val Tyr Ala Arg Arg Phe Phe Asp Arg Phe Ser Pro Asp Phe 195 200 205 Lys Asp Phe His Gly Ser Glu He Asn Arg Leu Phe Ser Val Asn Ser 210 215 220 He Asp His He Lys Glu Asn Glu Val Ala Ser Ala Phe Gin Ser His 225 230 235 240 Lys Tyr Arg He Thr Met Ser Lys Thr Thr Leu Asn Leu Leu Leu Tyr 245 250 255 Phe Leu Asn Glu Asn Glu Ser He Gly Gly Ser Leu He He Ser Val 260 265 270 He Asn Gin His Leu Asp Pro Asn He Val Glu Ser Val Thr Ala Arg 275 280 285 Glu Lys Leu Ala Asp Gly He Lys Val Leu Ser Asp Ser Glu Asn Gly 290 295 300 Asn G s Gin Asn Leu Glu t Asn Ser Val Pro Val Lys Leu Gly 305 310 315 ._ ) Pro Phe Pro Lys Asp Glu Glu Phe Val Lys Glu He Glu Thr Glu Leu 325 330 335 Lys He Lys Asp A Gin Glu Lys Gin Lt sn Gin Gin Thr Ala Gly 340 345 350 Asp Asn Tyr Ser Gly Ala Asn Asn Arg Thr Leu Leu Gin Glu Tyr Lys 355 360 365 Ala Met Asn Asn Glu Lys Phe Lys Asp Asn Thr Gly Asp Asp Asp Lys 370 375 380 Asp Lys He Lys Asp Lys He Ala Lys Asp Glu Glu Lys Lys Glu Ser 385 390 395 400 Glu Leu Lys Val Asp Gly Glu Lys Lys Asp Ser Asn Leu Ser Ser Pro 405 410 415 Ala Arg Asp He Leu Pro Leu Pro Pro Lys Thr Ala Leu Asp Leu Lys 420 425 430 Leu Glu He Gin Lys Val Lys Glu Ser Arg Asp Ala He Lys Leu Asp 435 440 445 Asn Leu Gin Leu Ala Leu Pro Ser Val Cys Met Tyr Thr Phe Gin Asn 450 455 460 Thr Asn Lys Asp Met Ser Cys Leu Asp Phe Ser Asp Asp Cys Arg He 465 470 475 480 Ala Ala Ala Gly Phe Gin Asp Ser Tyr He Lys He Tφ Ser Leu Asp 485 490 495 Gly Ser Ser Leu Asn Asn Pro Asn He Ala Leu Asn Asn Asn Asp Lys 500 505 510 Asp Glu Asp Pro Thr Cys Lys Thr Leu Val Gly His Ser Gly Thr Val 515 520 525 Tyr Ser Thr Ser Phe Ser Pro Asp Asn Lys Tyr Leu Leu Ser Gly Ser 530 535 540 Glu Asp Lys Thr Val Arg Leu Trp Ser Met Asp Thr His Thr Ala Leu 545 550 555 560 Val Ser Tyr Lys Gly His Asn His Pro Val Tφ Asp Val Ser Phe Ser 565 570 575 Pro Leu Gly His Tyr Phe Ala Thr Ala Ser His Asp Gin Thr Ala Arg 580 585 590 Leu Tφ Ser Cys Asp His He Tyr Pro Leu Arg He Phe Ala Gly His 595 600 605 Leu Asn Asp Val Asp Cys Val Ser Phe His Pro Asn Gly Cys Tyr Val 610 615 620 Phe Thr Gly Ser Ser Asp Lys Thr Cys Arg Met Tφ Asp Val Ser Thr 625 630 635 640 Gly Asp Ser Val Arg Leu Phe Leu Gly His Thr Ala Pro Val He Ser 645 650 655 He Ala Val Cys Pro Asp Gly Arg Tφ Leu Ser Thr Gly Ser Glu Asp 660 665 670 Gly He He Asn Val Tφ Asp He Gly Thr Gly Lys Arg Leu Lys Gin 675 680 685 Met Arg Gly His Gly Lys Asn Ala He Tyr Ser Leu Ser Tyr Ser Lys 690 695 700 Glu Gly Asn Val Leu He Ser Gly Gly Ala Asp His Thr Val Arg Val 705 710 715 720 SUBSTITUTE MET (RULE 26) Tφ Asp Leu Lys Lys Ala Thr Thr Glu Pro Ser Ala Glu Pro Asp Glu 725 730 735 Pro Phe He Gly Tyr Leu Gly Asp Val Thr Ala Ser He Asn Gin Asp 740 745 750 He Lys Glu Tyr Gly Arg Arg Arg Thr Val He Pro Thr Ser Asp Leu 755 760 765 Val Ala Ser Phe Tyr Thr Lys Lys Thr Pro Val Phe Lys Val Lys Phe 770 775 780 Ser Arg Ser Asn Leu Ala Leu Ala Gly Gly Ala Phe Arg Pro 785 790 795 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 704 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: D. melanogaster (vii) IMMEDIATE SOURCE: (B) CLONE: TAFII-80 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Ser Leu Glu Val Ser Asn He Asn Gly Gly Asn Gly Thr Gin Leu 1 5 10 15 Ser His Asp Lys Arg Glu Leu Leu Cys Leu Leu Lys Leu He Lys Lys 20 25 30 Tyr Gin Leu Lys Ser Thr Glu Glu Leu Leu Cys Gin Glu Ala Asn Val 35 40 45 Ser Ser Val Glu Leu Ser Glu He Ser Glu Ser Asp Val Gin Gin Val 50 55 60 Leu Gly Ala Val Leu Gly Ala Gly Asp Ala Asn Arg Glu Arg Lys H' - 65 70 75 80 Val Gin Ser Pro Ala Gin Gly His Lys Gin Ser Ala Val Thr Glu Ala 85 90 95 Asn Ala Ala Glu Glu Leu Ala Lys Phe He Asp Asp Asp Ser Phe Asp 100 105 1 10 Ala Gin His Tyr Glu Gin Ala Tyr Lys Glu Leu Arg Thr Phe Val Glu 1 15 120 125 Asp Ser Leu Asp He Tyr Lys His Glu Leu Ser Met Val Leu Tyr Pro 130 135 140 He Leu Val Gin He Tyr Phe Lys He Leu Ala Ser Gly Leu Arg Glu 145 150 155 160 Lys Ala Lys Glu Phe He Glu Lys Tyr Lys Cys Asp Leu Asp Gly Tyr 165 170 175 Tyr He Glu Gly Leu Phe Asn Leu Leu Leu Leu Ser Lys Pro Glu Glu 180 185 190 Leu Leu Glu Asn Asp Leu Val Val Ala Met Glu Gin Asp Lys Phe Val 195 200 205 He Arg Met Ser Arg Asp Ser His Ser Leu Phe Lys Arg His He Gin 210 215 220 Asp Arg Arg Gin Glu Val Val Ala Asp He Val Ser Lys Tyr Leu His 225 230 235 240 Phe Asp Thr Tyr Glu Gly Met Ala Arg Asn Lys Leu Gin Cys Val Ala 245 250 255 Thr Ala Gly Ser His Leu Gly Glu Ala Lys Arg Gin Asp Asn Lys Met 260 265 270 Arg Val Tyr Tyr Gly Leu Leu Lys Glu Val Asp Phe Gin Thr Leu Thr 275 280 285 Thr Pro Ala Pro Ala Pro Glu Glu Glu Asp Asp Asp Pro Asp Ala Pro 290 295 300 Asp Arg Pro Lys Lys Lys Lys Pro Lys Lys Asp Pro Leu Leu Ser Lys 305 310 315 320 Lys Ser Lys Ser Asp Pro Asn Ala Pro Ser He Asp Arg He Pro Leu 325 330 335 Pro Glu Leu Lys Asp Ser Asp Lys Leu Leu Lys Leu Lys Ala Leu Ar 340 345 350 Glu Ala Ser Lys Arg Leu Ala Leu Ser Lys Asp Gin Leu Pro Ser Ala 355 360 365 Val Phe Tyr Thr Val Leu Asn Ser His Gin Gly Val Thr Cys Ala Glu 370 375 380 He Ser Asp Asp Ser Thr Met Leu Ala Cys Gly Phe Gly Asp Ser Ser 385 390 395 400 Val Arg He Tφ Ser Leu Thr Pro Ala Asn Val Arg Thr Leu Lys Asp 405 410 415 Ala Asp Ser Leu Arg Glu ' _u Asp Lys Glu Ser Ala Asp He Asn Val 420 425 430 Arg Met Leu Asp Asp Arg Ser Gly Glu Val Thr Arg S Leu Met Gly 435 440 445 His Thr Gly Pro Val Tyr Arg Cys Ala Phe Ala Pro Glu Met Asn Leu 450 455 460 Leu Leu Ser Cys Ser Glu Asp Ser Thr He Arg Leu Tφ Ser Leu Leu 465 470 475 480 Thr Tφ Ser Cys Val Val Thr Tyr Arg Gly His Val Tyr Pro Val Tφ 485 490 4°5 Asp Val Arg Phe Ala Pro His Gly Tyr Tyr Phe Val Ser Cys Ser Tyr 500 505 510 Asp Lys Thr Ala Arg Leu Tφ Ala Thr Asp Ser Asn Gin Ala Leu Arg 515 520 525 Val Phe Val Gly His Leu Ser Asp Val Asp Cys Val Gin Phe His Pro 530 535 540 Asn Ser Asn Tyr Val Ala Thr Gly Ser Ser Asp Arg Thr Val Arg Leu 545 550 555 560 Tφ Asp Asn Met Thr Gly Gin Ser Val Arg Leu Met Thr Gly His Lys 565 570 575 Gly Ser Val Ser Ser Leu Ala Phe Ser Ala Cys Gly Arg Tyr Leu Ala 580 585 590 Ser Gly Ser Val Asp His Asn He He He Tφ Asp Leu Ser Asn Gly 595 600 605 Ser Leu Val Thr Thr Leu Leu Arg His Thr Ser Thr Val Thr Thr He 610 615 620 Thr Phe Ser Arg Asp Gly Thr Val Leu Ala Ala Ala Gly Leu Asp Asn 625 630 635 640 Asn Leu Thr Leu Tφ Asp Phe His Lys Val Thr Glu Asp Tyr He Ser 645 650 655 Asn His He Thr Val Ser His His Gin Asp Glu Asn Asp Glu Asp Val 660 665 670 Tyr Leu Met Arg Thr Phe Pro Ser Lys Asn Ser Pro Phe Val Ser Leu 675 680 685 His Phe Thr Arg Arg Asn Leu Leu Met Cys Val Gly Leu Phe Lys Ser 690 695 700 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1377 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: D. melanogaster (vii) IMMEDIATE SOURCE: (B) CLONE: TAFII-250 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Glu Ser Asp Asn Ser Asp Asp Glu Gly Ser He Gly Asn Gly Leu 1 5 10 15 Asp Leu Thr Gly He Leu Phe Gly Asn He Asp Ser Glu Gly Arg Leu 20 25 30 Leu Gin Asp Asp Asp Gly Glu Gly Arg Gly Gly Thr Gly Phe Asp Ala 35 40 45 Glu Leu Arg Glu Asn He Gly Ser Leu Ser Lys Leu Gly Leu Asp Ser 50 55 60 Met Leu Leu Glu Val He Asp Leu Lys Glu Ala Glu Pro Pro Ser Asp 65 70 75 80 Asp Glu Glu Glu Glu Asp Ala Arg Pro Ser Ala Val Ser Ala Ser Gly 85 90 95 Gly Met Ser Ala Phe Asp Ala Leu Lys Ala Gly Val Lys Arg Glu Glu 100 105 1 10 Arg Glu Asp Gly Ala Val Lys Ala Gin Asp Asp Ala He Asp Tyr Ser 1 15 120 125 Asp He Thr Glu Leu Ser Glu Asp Cys Pro Arg Thr Pro Pro Glu Glu 130 135 140 Thr Ser Thr Tyr Asp Asp Leu Glu Asp Ala He Pro Ala Ser Lys Val 145 150 155 160 Glu Ala Lys Leu Thr Lys Asp Asp Lys Glu Leu Met Pro Pro Pro Ser 165 170 175 Ala Pro Met Arg Ser Gly Ser Gly Gly Gly He Glu Glu Pro Ala Lys 180 185 190 Ser Asn Asp Ala Ser Ser Pro Ser Asp Asp Ser Lys Ser Thr Asp Ser 195 200 205 Lys Asp Ala Asp Arg Lys Leu Asp Thr Pro Leu Ala Asp He Leu Pro 210 215 220 Ser Lys Tyr Gin Asn Val Asp Val Arg Glu Leu Phe Pro Asp Phe Arg 225 230 235 240 Pro Gin Lys Val Leu Arg Phe Ser Arg Leu Phe Gly Pro Gly Lys Pro 245 250 255 Thr Ser Leu Pro Gin He Tφ Arg Glu Val Arg Lys Arg Arg Arg Lys 260 265 270 Arg Asn Gin Ser Arg Asp Gin Lys Thr Thr Asn Thr Gly Gly Ser Asp 275 280 285 Ser Pro Ser Asp Thr Glu Glu Pro Arg Lys Arg Gly Phe Ser Leu His 290 295 300 Tyr Ala Ala Glu Pro Thr Pro Ala Glu Cys Met Ser Asp Asp Glu Asp 305 310 315 320 Lys Leu Leu Gly Asp Phe His Ser Glu Asp Val Arg Pro Phe Gly Pro 325 330 335 Asp Asn Gly Glu Asn Ser Asp Gin Lys Pro Lys Val Ala Asp Trp Arg 340 345 350 Phe Gly Pro Ala Gin He Tφ Tyr Asp He Leu Glu Val Pro Asp Ser 355 360 365 Gly Glu Gly Phe Asn Tyr Gly Phe Lys Thr Lys Ala Ala Ser Thr Ser 370 375 380 Ser Gin Gin Gin Leu Lys Asp Glu Arg Arg Val Lys Ser Pro Glu Asp 385 390 395 400 Asp Val Glu Asp Pro Ser He Ala Asp Asp Ala Phe Leu Met Val Ser 405 410 415 Gin Leu His Tφ Glu Asp Asp Val Val Tφ Asp Gly Asn Asp He Lys 420 425 430 Ala Lys Val Leu Gin Lys Leu Asn Ser Lys Thr Asn Ala Ala Gly Tφ 435 440 445 Leu Pro Ser Ser Gly Ser Arg Thr Ala Gly Ala Phe Ser Gin Pro Gly 450 455 460 Lys Pro Ser Met Pro Val Gly Ser Gly Ser Ser Lys Gin Gly Ser Gly 465 470 475 480 Ala Ser Ser Lys Lys Ala Gin Gin Asn Ala Gin Ala Lys Pro Ala Glu 485 490 495 Ala Pro Asp Asp Thr Tφ Tyr Ser Leu Phe Pro Val Glu Asn Glu Glu 500 505 510 Leu He Tyr Tyr Lys Tφ Glu Asp Glu Val He Tφ Asp Ala Gin Gin 515 520 525 Val Ser Lys Val Pro Lys Pro Lys Val Leu Thr Leu Asp Pro Asn Asp 530 535 540 Glu Asn He He Leu Gly He Pro Asp Asp He Asp Pro Ser Lys He 545 550 555 560 Asn Lys Ser Thr Gly Pro Pro Pro Lys He Lys He Pro His Pro His 565 570 575 Val Lys Lys Ser Lys He Leu Leu Gly Lys Ala Gly Val He Asn Val 580 585 590 Leu Ala Glu Asp Thr Pro Pro Pro Pro Pro Lys Ser Pro Asp Arg Asp 595 600 605 Pro Phe Asn He Ser Asn Asp Thr Tyr Tyr Thr Pro Lys Thr Glu Pro 610 615 620 Thr Leu Arg Leu Lys Val Gly Gly Asn Leu He Gin His Ser Thr Pro 625 630 635 640 Val Val Glu Leu Arg Ala Pro Phe Val Pro Thr Arg Met Gly Pro Met 645 650 655 Asn Val Arg Ala Phe His Arg Pro Pro Leu Lys Lys Tyr Ser His Gly 660 665 670 Pro Met Ala Gin Ser He Pro His Pro Val Thr Pro Leu Leu Lys Thr 675 680 685 He Ala Lys Lys Ala Lys Gin Arg Glu Val Glu Arg He Ala Ser Gly 690 695 700 Gly Gly Asp Val Phe Phe Met Arg Asn Pro Glu Asp Leu Ser Gly Arg 705 710 715 720 Asp Gly Asp He Val Leu Ala Glu Phe Cys Glu Glu His Pro Pro Leu 725 730 735 He Asn Gin Val Gly Met Cys Ser Lys He Lys Asn Tyr Tyr Lys Arg 740 745 750 Lys Ala Glu Lys Asp Ser Gly Pro Gin Asp Tyr Val Tyr Gly Glu Val 755 760 765 Ala Phe Ala Arg Thr Ser Pro Phe Leu Gly He Leu His Pro Gly Gin 770 775 780 Cys He Gin Ala He Glu Asn Asn Met Tyr Arg Ala Pro He Tyr Pro 785 790 795 800 His Lys Met Ala His Asn Asp Phe Leu Val He Arg Thr Arg Asn His 805 810 815 Tyr Tφ He Arg Ser Val Asn Ser He Tyr Thr Val Gly Gin Glu Cys 820 825 830 Pro Leu Tyr Glu Val Pro Gly Pro Asn Ser Lys Arg Ala Asn Asn Phe 835 840 845 Thr Arg Asp Phe Leu Gin Val Thr He Tyr Arg Leu Phe Tφ Lys Ser 850 855 860 Arg Asp Asn Pro Arg Arg He Arg Met Asp Asp He Lys Gin Ala Phe 865 870 875 880 Pro Ala His Ser Glu Ser Ser He Arg Lys Arg Leu Lys Gin Cys Ala 885 890 895 Asp Phe Lys Arg Thr Gly Met Asp Ser Asn Trp Tφ Val He Lys Pro 900 905 910 Glu Phe Arg Leu Pro Ser Glu Glu Glu He Arg Ala Met Val Ser Pro 915 920 925 Glu Gin Cys Cys Ala Tyr Phe Ser Met He Ala Ala Glu Gin Arg Leu 930 935 940 Lys Asp Ala Gly Tyr Gly Glu Lys Phe Leu Phe Ala Pro Gin Glu Asp 945 950 955 960 Asp Asp Glu Glu Ala Gin Leu Lys Leu Asp Asp Glu Val Lys Val Ala 965 970 975 Pro Tφ Asn Thr Thr Arg Ala Tyr He Gin Ala Met Arg Gly Lys Cys 980 985 990 Leu Leu Gin Leu Ser Gly Pro Ala Asp Pro Thr Gly Cys Gly Glu Gly 995 1000 1005 Phe Ser Tyr Val Arg Val Pro Asn Lys Pro Thr Gin Thr Lys Glu Glu 1010 1015 1020 Gin Glu Ser Gin Pro Lys Arg Ser Val Thr Gly Thr Asp Ala Asp Leu 1025 1030 1035 1040 Arg Arg Leu Pro Leu Gin Arg Ala Lys Glu Leu Leu Arg Gin Phe Lys 1045 1050 1055 Val Pro Glu Glu Glu He Lys Lys Leu Ser Arg Tφ Glu Val He Asp 1060 1065 1070 Val Val Arg Thr Leu Ser Thr Glu Lys Ala Lys Ala Gly Glu Glu Gly 1075 1080 1085 Met Asp Lys Phe Ser Arg Gly Asn Arg Phe Ser He Ala Glu His Gin 1090 1095 1 100 Glu Arg Tyr Lys Glu Glu Cys Gin Arg He Phe Asp Leu Gin Asn Arg 1 105 1 1 10 1 1 15 1 120 Val Leu Ala Ser Ser Glu Val Leu Ser Thr Asp Glu Ala Glu Ser Ser 1 125 1 130 1 135 Ala Ser Glu Glu Ser Asp Leu Glu Glu Leu Gly Lys Asn Leu Glu Asn 1140 1 145 1150 Met Leu Ser Asn Lys Lys Thr Ser Thr Gin Leu Ser Arg Glu Arg Glu 1 155 1160 1165 Glu Leu Glu Arg Gin Glu Leu Leu Arg Gin Leu Asp Glu Glu His Gly 1 170 1 175 1 180 Gly Pro Ser Gly Ser Gly Gly Ala Lys Gly Ala Lys Gly Lys Asp Asp 1 185 1 190 1195 1200 Pro Gly Gin Gin Met Leu Ala Thr Asn Asn Gin Gly Arg He Leu Arg 1205 1210 1215 He Thr Arg Thr Phe Arg Gly Asn Asp Gly Lys Glu Tyr Thr Arg Val 1220 1225 1230 Glu Thr Val Arg Arg Gin Pro Val He Asp Ala Tyr He Lys He Arg 1235 1240 1245 SUBSTITUTE SHEET (RULE 261 Thr Thr Lys Asp Glu Gin Phe He Lys Gin Phe Ala Thr Leu Asp Glu 1250 1255 1260 Gin Gin Lys Glu Glu Met Lys Arg Glu Lys Arg Arg He Gin Glu Gin 1265 1270 1275 1280 Leu Arg Arg He Lys Arg Asn Gin Glu Arg Glu Arg Leu Ala Gin Leu 1285 1290 1295 Ala Gin Asn Gin Lys Leu Gin Pro Gly Gly Met Pro Thr Ser Leu Gly 1300 1305 1310 Asp Pro Lys Ser Ser Gly Gly His Ser His Lys Glu Arg Asp Ser Gly 1315 1320 1325 Tyr ys Glu Val Ser Pro Ser Arg Lys Lys Phe Lys Leu Lys Pro Asp 1330 1335 1340 Leu Lys Leu Lys Cys Gly Ala Cys Gly Gin Val Gly His Met Arg Thr 1345 1350 1355 1360 Asn Lys Ala Cys Pro Leu Tyr Ser Gly Met Gin Ser Ser Leu Ser Gin 1365 1370 1375 Ser (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid SUBSTITUTE SHEET (RULE 26V (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: S. cerevisiae (vii) IMMEDIATE SOURCE: (B) CLONE: TAF-90 PEPTIDE 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Ala Thr Thr Glu Pro Ser Ala Glu Pro Asp Glu Pro Phe He Gly Tyr 1 5 10 15 Leu Gly Asp Val Thr Ala 20 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (vi) ORIGINAL SOURCE: (A) ORGANISM: S. cerevisiae (vii) IMMEDIATE SOURCE: (B) CLONE: TAF-90 PRIMER 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AGATACTTGA AAATCTAGAA TGTCACAC 28 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (vi) ORIGINAL SOURCE: (A) ORGANISM: S. cerevisiae (vii) IMMEDIATE SOURCE: (B) CLONE: TAF-90 PRIMER 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGTTATACTT ATTACATATC ACTTCCATGC 30 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: S. cerevisiae (vii) IMMEDIATE SOURCE: (B) CLONE: TAF- 145 PEPTIDE 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Asn He Asn His Leu Phe Thr Val Gly Gin Thr Phe Pro Val Glu Glu 1 5 10 15 He Pro Gly Pro Asn 20 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: S. cerevisiae (vii) IMMEDIATE SOURCE: (B) CLONE: TAF- 145 PEPTIDE 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Leu Pro Val Gly Glu Thr His Val Leu Gly Val Gin Asp Lys Ser Pro 1 5 10 15 Phe (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26 (ii) MOLECULE TYPE: cDNA to mRNA (vi) ORIGINAL SOURCE: (A) ORGANISM: S. cerevisiae (vii) IMMEDIATE SOURCE: (B) CLONE: TAF-145 PRIMER 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TTYCCNGTNG ARGARAT 17 (2) INFORMATION FOR SEQ ID NO: l 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (vi) ORIGINAL SOURCE: (A) ORGANISM: S. cerevisiae (vii) IMMEDIATE SOURCE: (B) CLONE: TAF- 145 PRIMER 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l l : GTNGGNGARA CNCAYGT 17

Claims: WHAT IS CLAIMED IS: 1. An isolated nuclear polypeptide having the sequence as set forth in Figure 6B SEQ. I.D. No. 1. 2. A fragment of the polypeptide of claim 1 which inhibits the interaction of said polypeptide and TATA-box Binding Protein (TBP). 3. A complex isolated from a fungus, said complex comprising a plurality of polypeptides, said polypeptides having molecular masses of about 180 kDa, 145 kDa, 1 16 kDa, 90 kDa, 68 kDa, 51-54 kDa, 47 kDa, and 30 kDa. respectively, and said complex activating gene iranscription of said fungus. 4. The complex of claim 3, wherein said complex is derived from a fungal species selected from the group consisting of S. cerevisiae, C. albicans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, and OAspergillus fumigatus. 5. A method for inhibiting fungal transcription in a fungal cell. comprising contacting said cell with an agent that selectively interferes with the transcriptional activation activity of the complex of claim 3. 6. An isolated nucleic acid selected from the group consisting of DNA encoding the polypeptide sequence set forth in Figure 6B and sequences that hybridize thereto under stringent hybridization conditions. 7. A DNA vector comprising the DNA sequence encoding the polypeptide sequence set forth in Figure 6B SEQ. I.D. No. 1 operably linked to a transcription regulatory element. 8. The polypeptide of claim 1. wherein said polypeptide is recombinantly produced. 64 0342/2A404 9. A cell comprising the DNA vector of claim 7. 10. A process for the production of recombinant TAF- 145 comprising (i) culturing the cell of claim 9 under conditions suitable for the expression of the TAF- 145 polypeptide, and (ii) recovering said polypeptide from said cell. 11. A method for screening a plurality of test compounds to identify an agent useful in the treatment of fungal diseases in mammals which comprises: (i) exposing the complex of claim 3 or polypeptides derived from said complex to TATA-box Binding Protein (TBP) in the presence of at least one test compound, and (ii) identifying those test compounds that inhibit the binding of said TBP to said complex or said polypeptides. 12. The method of claim 11 which comprises: (i) exposing the polypeptide of claim 1 to TATA-box Binding Protein (TBP) in the presence of at least one test compound, and (ϋ) identifying those test compounds that inhibit the binding of said TBP to said polypeptide. 13. The method of claim 11 further comprising immobilizing said polypeptide on a solid surface. 14. A method for treating a fungal infection in a mammal in need of such treatment, which comprises administering to said mammal effective amounts for treating fungal infection of the pharmacological agent identified by the method of claim 11.

Inventors: GREEN MICHAEL D, REESE JOSEPH C, GREEN, MICHAEL, D., REESE, JOSEPH, C.
Applicants: UNIV MASSACHUSETTS MEDICAL, UNIVERSITY OF MASSACHUSETTS MEDICAL CENTER
Application year: 1995
Main classification: C12N15
IPC codes: A61K38, A61P31, C07K14, C12N1, C12N15, C12P21, C12Q1, C12R1