Specific Interactions and Potential Functions of Human TAFII100*

(Received for publication, October 7, 1996, and in revised form, December 19, 1996)

Yong Tao Dagger , Mohamed Guermah Dagger , Ernest Martinez Dagger , Thomas Oelgeschläger Dagger , Satoshi Hasegawa Dagger §, Ritsuko Takada Dagger , Tohru Yamamoto Dagger §, Masami Horikoshi Dagger and Robert G. Roeder Dagger par

From the Dagger  Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021 and the § Laboratory of Developmental Biology, Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
Note Added in Proof
REFERENCES


ABSTRACT

Human transcription initiation factor TFIID contains the TATA-binding protein (TBP) and several TBP-associated factors (TAFs). To investigate the structural organization and function of TFIID, we have cloned and expressed a cDNA encoding the third largest human TFIID subunit, hTAFII100. Immunoprecipitation studies demonstrate that hTAFII100 is an integral subunit that is associated with all transcriptionally-competent forms of TFIID. They further suggest that at least part of the N-terminal region lies on the surface of TFIID, while a C-terminal region containing conserved WD-40 repeats appears inaccessible. Both in vivo and in vitro assays indicate that hTAFII100 interacts strongly with the histone H4-related hTAFII80 and the histone H3-related hTAFII31, as well as a stable complex comprised of both hTAFII80 and hTAFII31. Apparently weaker interactions of hTAFII100 with TBP, hTAFII250, hTAFII28, and hTAFII20, but not hTAFII55, also have been observed. These results suggest a role for hTAFII100 in stabilizing interactions of TAFs, especially the histone-like TAFs, in TFIID. In addition, functional studies show that anti-hTAFII100 antibodies selectively inhibit basal transcription from a TATA-less initiator-containing promoter, relative to a TATA-containing promoter, suggesting a possible core promoter-specific function for hTAFII100.


INTRODUCTION

TFIID is a general transcription initiation factor comprised of a small TATA-binding polypeptide (TBP)1 and a large number of TBP-associated factors (TAFs), all of which are highly conserved in evolution (reviewed in Ref. 1). One major function of TFIID is to recognize common core promoter elements and to nucleate assembly of other general factors (TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH) and RNA polymerase II into a functional preinitiation complex (reviewed in Ref. 2). The primary interaction with core promoters containing TATA elements is through TBP, which is sufficient to mediate basal transcription in the absence of TAFs; in contrast, primary interactions with core promoters lacking TATA elements is thought to involve recognition of alternative core promoter elements (e.g. initiator elements) by TAFs and/or other factors that also are essential for basal transcription from these promoters (reviewed in Ref. 2).

In vitro studies in human and Drosophila systems have shown an essential role in activation both for TAFs and for other cofactors (reviewed in Refs. 1-4). Consistent with earlier studies showing that activators have qualitative and quantitative effects on TFIID binding that correlate with enhanced recruitment and function of other general factors (reviewed in Ref. 2), more recent studies have described specific activator-TAF interactions that are relevant to the overall function of the activators in vitro (reviewed in Refs. 1 and 3). More recently, and consistent with its role as a co-activator, TFIIA also has been shown to alter the topological arrangement of TAFs within a TFIID-promoter complex (5). These various studies have led to speculation that activator effects through TFIID may involve, in part, conformational changes that either stabilize TFIID binding on the promoter or facilitate interactions of other general initiation factors with the TFIID-promoter complex (reviewed in Ref. 2). Although recent studies in yeast have questioned a completely general role for TAFs in activator-mediated transcription in vivo (6, 7), the yeast TAFs are essential for viability and are likely to be necessary for activation of at least some genes and, potentially, for other events important for cell cycle progression. Possibly related to the latter point, a fraction of cellular TFIID has been shown to remain associated with chromosomes during mitosis in human cells, and mitosis-specific phosphorylation of TAFs has been shown to inactivate activator-specific functions of TFIID (8).

Of key importance for an understanding of the various TFIID functions is an appreciation of the overall structure of TFIID, including both the primary sequences and structures of individual subunits and their interactions and topological organization within TFIID. The cloning of cDNAs encoding most of the yeast, Drosophila, and human TAFs has provided relevant information, including schemes for a number of protein-protein interactions (9-13, reviewed in Ref. 1). Also of note is the documentation of three histone-like TAFs and the indication, from biochemical (10, 13-15) and structural (16) studies, of a histone-like octamer (in TFIID) comprised of the H4-like (hTAFII80/dTAFII62), H3-like (hTAFII31/dTAFII42), and H2B-like (hTAFII20/dTAFII28) TAFs.

Here we document the cloning of a cDNA encoding hTAFII100, the presence of hTAFII100 within all functional TFIID molecules, novel interactions of hTAFII100 with other TFIID subunits, and a possible core promoter-specific function.


EXPERIMENTAL PROCEDURES

Isolation of cDNA Clones for hTAFII100

TFIID was affinity-purified from a phosphocellulose (Whatman P11) fraction (0.85 M KCl) of HeLa nuclear extract by use of an anti-TBP antibody (17). TFIID subunits were resolved by SDS-PAGE and transferred to a poly(vinylidene difluoride) membrane. The 100-kDa polypeptide was excised and digested with endoproteinase Lys-C (18). Sequence analysis of the purified peptides yielded the following peptide sequences: EAEEALRREAXLLEEA, PEIEVPLDDEXEEG, and VAVEDQPDVXAXL. Two degenerate oligonucleotides, 5'-GAIGCIGA(A/G)GA(A/G)GCICTICGICGIGA(A/G)GCIIIICTICTIGAIGAIGC-3' and 5'-CCIGA(A/G)ATIGA(A/G)GTICCICTIGATGATGA(A/G)IIIGAIGAIGG-3' were synthesized and used to screen both HeLa and Namalwa cDNA libraries according to standard procedures (19). Three overlapping cDNA clones were isolated. The cDNAs were sequenced entirely on both strands by use of Sequenase (U. S. Biochemical Corp.).

Expression and Purification of Recombinant TAFs

The plasmids which contain cDNAs encoding hemagglutinin (HA)-hTAFII250 and FLAG-hTAFII55 have been described (20). Expression plasmids for HA- or FLAG-hTAFII80, His-TBP, FLAG-hTAFII31, FLAG-hTAFII28, and HA-hTAFII20 were constructed by polymerase chain reaction, creating, in each case, an NdeI site at the N-terminal end and an appropriate restriction enzyme site at the C-terminal end following the natural stop codon. The large number of primers used in the PCR reactions has precluded description of their exact sequences, but the information is available upon request. The PCR-generated fragments were then inserted into pFLAG(AS)-7 or HA-pGEM7 at NdeI and appropriate sites (Ref. 13).2 The cDNAs encoding epitope-tagged constructs were then inserted into pVL1393/2 (Invitrogen) as described (20). For each TFIID subunit, an individual recombinant baculovirus was generated by co-transfecting corresponding cDNA and BaculoGold linearized baculovirus DNA (PharMingen) into Sf9 insect cells with cationic liposomes (Invitrogen). Each recombinant baculovirus was further amplified by repeated infection of Sf9 cells (3-4 times). For production of recombinant TAF proteins, Sf9 cells were infected by the amplified baculovirus, harvested after 48 h, and sonicated in 0.05 volume of Buffer C (20 mM Tris, 0.2 mM EDTA, 20% glycerol, 1 mM dithiothreitol, pH 7.9) containing 100 mM KCl and 0.5 mM phenylmethylsulfonyl fluoride. After removal of insoluble debris by centrifugation, supernatants were subjected to affinity purification on anti-FLAG antibody (M2-agarose, Kodak) or anti-HA antibody (12CA5 monoclonal antibody) columns. Epitope-tagged recombinant proteins were eluted with FLAG- or HA-peptides (20) and further purified by one or two steps of ion exchange chromatography. The recombinant proteins were more than 90% pure as judged by SDS-PAGE and silver staining.

Expression of hTAFII100 and Its Derivatives

A PCR-based method was used to create an NdeI site overlapping the initiation codon. The PCR product was inserted into pFLAG(AS)-7 and then subcloned into the baculovirus vector pVL1393 as described (20). Full-length recombinant hTAFII100 was expressed in Sf9 cells as described above. For expression of histidine-tag and GST fusion proteins, the cDNA sequences encoding N-terminal (1-455) and C-terminal (456-750) portions of hTAFII100 were amplified by PCR (creating in each case an NdeI site at the N terminus and a BamHI site following a stop codon at the C terminus). PCR-generated fragments were then inserted into bacterial expression vectors at NdeI and BamHI sites (for the histidine-tag, 6HisT-pRSET was used; for the GST fusion, pGEX-2TL(+) was used (13)). His-tagged hTAFII100(1-455) and His-tagged hTAFII100(456-750) were produced in Escherichia coli as described (13). The recombinant proteins were purified by chromatography on Ni-NTA-agarose (Qiagen, Chatsworth, CA). GST fusion proteins were generated in E. coli and purified on glutathione-Sepharose columns (Pharmacia Biotech Inc.). A histidine-tagged full-length hTAFII100 (His-hTAFII100) cDNA construct was similar to the FLAG-tagged construct, except for the use of the 6HisT-pRSET vector in place of pFLAG(AS)-7. His-hTAFII100 was generated in the Promega TNT reticulocyte lysate system and labeled with [35S]methionine according to the manufacturer's instructions. After purification on a mini Ni-NTA-agarose column (20 µl), protein was stored at -80 °C until use.

Generation and Use of Antibodies against hTAFII100

His-tagged hTAFII100(1-455) and His-tagged hTAFII100(456-750) were purified as above and subjected to preparative SDS-PAGE. Gel slices containing the corresponding His-tagged proteins were crushed and used for immunization of two rabbits. The anti-hTAFII100 antibodies were then affinity-purified on columns containing either GST-hTAFII100(1-455) or GST-hTAFII100(456-800) proteins. For immunodepletion (Fig. 3) and immunoprecipitation (Fig. 4) experiments, 1-2 mg amounts of antibodies were further purified by binding to 1 ml of protein A-Sepharose (Pharmacia) and then cross-linked with dimethylpimelimidate as described (21). For the immunodepletion studies, 5 ml of nuclear extract was cycled through 2 ml of cross-linked anti-hTAFII100-protein A-Sepharose (2 mg/ml antibody) 5 times and the flow-through was used for Western blotting analysis and transcription assay. For analysis of direct inhibitory effects of antibodies on transcription (Fig. 5), anti-hTAFII100 antibody was antigen-affinity-purified (as above), bound to and eluted from Protein A-Sepharose, and dialyzed against Buffer C containing 100 mM KCl. Different amounts of antibody, as indicated in Fig. 5, were then incubated with 4-µl aliquots of nuclear extract at room temperature for 30 min. The extracts were employed directly, without antibody removal, in transcription assays.


Fig. 3. Quantitative immunodepletion of TFIID transcription activity by anti-hTAFII100 antibody. Antibodies to hTAFII100(1-455) and hTAFII100(456-750) designated alpha hTAFII100N and alpha hTAFII100C, respectively, were used to immunodeplete TFIID from HeLa nuclear extracts. A, extracts treated with alpha hTAFII100N (lane 3), alpha hTAFII100C (lane 2), and control IgG (lane 1) were subjected to SDS-PAGE and blots were probed with antibodies against hTAFII135, hTAFII100, hTAFII43, and TBP as indicated. B, extracts treated with control IgG (lanes 1-3), alpha hTAFII100C (lanes 4-6), and alpha hTAFII100N (lanes 7-9) were used for in vitro transcription assays with a template (pMLDelta 53) containing the adenovirus major late core promoter sequence (lower arrow) and a Gal4-VP16 responsive template (pG5HMC2AT) containing 5 Gal4-binding sites upstream of the core promoter (upper arrow). The volumes (µl) of Gal4VP16 activator (30 ng/µl) added to the reactions are indicated at the top. C, TFIID can restore the transcription activity of nuclear extracts depleted by anti-hTAFII100N antibody. In vitro transcription reactions containing control IgG treated (lanes 1-2) or anti-hTAFII100N antibody treated (lanes 3-8) nuclear extracts were supplemented with TFIID and activators (or buffer) as indicated at the top. GAL4-VP16 was used in lanes 2, 4, and 6, and GAL4p65(NFkappa B) was used in lane 8. D, the alpha hTAFII100N immunoprecipitate from HeLa nuclear extract was analyzed by Western blot analysis with the indicated TBP and TAF antibody probes.
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Fig. 4. A, intracellular interactions of hTAFII100 with other subunits of TFIID. The baculovirus/Sf9 cell-based assays are described under "Experimental Procedures." Control inputs (I) and anti-hTAFII100 immunoprecipitates (B) from Sf9 cell extracts containing individual TFIID subunits expressed in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of FLAG-hTAFII100 were analyzed by SDS-PAGE and immunoblotted with antibodies to TFIID subunits as indicated. Input samples contained 10% of the amounts used for the immunoprecipitation. B, in vitro interactions of hTAFII100 with highly purified TFIID subunits. Purified recombinant TBP and TAFs were immunoprecipitated by anti-hTAFII100 antibodies following incubation in the presence (lane 3) or absence (lane 2) of FLAG-hTAFII100. Immunoprecipitates (lanes 2 and 3) and 10% of each input (lane 1) were analyzed by SDS-PAGE and immunoblotting with antibodies to TFIID subunits as in A. C, silver staining of FLAG-hTAFII80, FLAG-hTAFII31, and a preformed FLAG-hTAFII80·hTAFII31 complex. Sf9 cell extracts containing expressed FLAG-hTAFII31 (lane 1), FLAG-hTAFII80 (lane 2), and both FLAG-hTAFII80 and hTAFII31 (lane 3) were prepared using buffer C (with 300 mM KCl). Recombinant proteins were bound to M2-agarose and, after extensive washing, eluted with FLAG peptide. Eluates were analyzed by SDS-PAGE and visualized by silver staining. D, direct interactions of hTAFII100 with hTAFII80, hTAFII31, and an hTAFII80·hTAFII31 complex. Purified hTAFII31, hTAFII80, and hTAFII80·hTAFII31 complex were incubated with purified 35S-labeled His-hTAFII100 and then subjected to immunoprecipitation by the antibodies indicated at the top. Immunoprecipitates were resolved by 6% SDS-PAGE and subjected to autoradiography. E, silver staining of SDS-PAGE resolved components from an hTAFII250·hTAFII100·TBP complex. This complex was assembled with purified TFIID subunits that were individually expressed via baculovirus vectors in Sf9 cells. Human hTAFII250 containing a fused N-terminal HA-epitope tag was immobilized on protein A-Sepharose containing covalently linked monoclonal antibodies directed against the HA-epitope. After extensive washing the beads were incubated (at 4 °C for 4 h) sequentially with molar excesses of TBP and hTAFII100. After each incubation unbound materials were removed by several washes. Finally, the complex was eluted with the HA peptide in Buffer C (with 100 mM KCl).
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Fig. 5. Specific inhibition of TATA-less promoter transcription by anti-hTAFII100N antibody. Effects of antibodies were tested by direct addition to nuclear extracts. A, effects on basal and activator-dependent transcription by anti-hTAFII100N antibody. The volumes (in µl) of GAL4-VP16 and antibody (1.6 mg/ml) are indicated at the top. The upper arrow indicates the transcripts from pG5HMC2AT and the lower arrow indicates the transcripts from pMLDelta 53. B, effects on transcription from the TATA-less TdT template by anti-hTAFII100N antibody. After incubation with different amounts of anti-hTAFII100N and anti-HA antibodies, HeLa nuclear extracts were employed for transcription from both Hsp70 and TdT templates. The primer extension products from the two templates are indicated by arrows. Lanes 2-4 contain 1, 1.4, and 1.8 µl of anti-hTAFII100N antibody (1.6 mg/ml), respectively, and lanes 5-7 contain 1, 1.4, and 1.8 µl of anti-HA antibody (1.6 mg/ml).
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In Vitro Transcription

Assays with the G-less cassette templates were carried out essentially as described (22). Apart from the normal buffer, substrates, and salts, reactions (25 µl) contained 100 ng of pG5HMC2AT and pMLDelta 53 supercoiled plasmid templates, 4-6 µl of HeLa nuclear extract and 30-60 ng of GAL4-VP16 or GAL4-p65(NFkappa B) as indicated. GAL4-VP16 and GAL4-p65 consist of amino acids 1-94 of the GAL4 DNA-binding domain fused, respectively, to a C-terminal portion (amino acids 412-490) of VP16 and a C-terminal portion (amino acids 416-550) of the p65 subunit of NFkappa B.2 Reactions were incubated at 30 °C for 60 min and the 32P-labeled RNA products were resolved by 6% PAGE with 8 M urea and visualized by autoradiography. Primer extension assays for Hsp70 and TdT transcription in nuclear extracts were carried out as described (23). The human 70-kDa heat shock protein (Hsp70) and murine terminal deoxynucleotidyltransferase (TdT) core promoter templates were, respectively, plasmids pHsp70((-33/+99-CAT) and pTdT(-41/+59) (24).

Intracellular Protein Interaction Assays

For each TFIID subunit, an individual recombinant baculovirus was generated as described above. Sf9 cells were co-infected by recombinant baculoviruses expressing FLAG-hTAFII100 and a second TFIID subunit and harvested after 48 h. The cell pellets were sonicated in 0.05 volume of Buffer C containing 100 mM KCl and 0.5 mM phenylmethylsulfonyl fluoride. After removal of insoluble debris by centrifugation, supernatants (cell lysates) were stored at -80 °C until used. For co-immunoprecipitation, in a 250-µl reaction, 200 µl of cell lysate were mixed with 10 µl of M2 beads (Kodak) or protein A-Sepharose cross-linked with anti-hTAFII100 antibodies, and the salt concentration was adjusted to 300 mM KCl and 0.2% Nonidet P-40. Binding assays were carried out at 4 °C for 2 h. The beads were washed 5 times with 1 ml of incubation buffer and boiled in 40 µl of SDS sample buffer. The eluates were analyzed by Western blot with specific antibodies against TAFs. In control experiments, cell lysates from singly infected cells lacking FLAG-hTAFII100 were used for co-immunoprecipitation.

In Vitro Protein Interaction Assays

For in vitro immunoprecipitation experiments, 40 ng of FLAG-hTAFII100 was immobilized on 10 µl of M2-agarose or protein A-Sepharose cross-linked with anti-hTAFII100 antibodies (for hTAFII250, FLAG-hTAFII55, and FLAG-hTAFII28). Binding reactions contained 100 µg of bovine serum albumin, 100 ng of recombinant TAFs (about 90% purity from Sf9 cells or E. coli), 300 mM KCl, and 0.2% Nonidet P-40 in a 200-µl volume. After incubating at 4 °C for 1 h, the beads were washed five times with 1 ml of incubation buffer and the proteins were eluted and analyzed by Western blotting.


RESULTS

Molecular Cloning of a Full-length Human cDNA Encoding TAFII100

The human TFIID complex was purified from a HeLa cell nuclear extract on a TBP antibody column (17). Two of the hTAFII100-derived peptide sequences, EAEEALRREAXLLEEA and PEIEVPLDDEXEEG, were used to design degenerate oligonucleotides to screen human cDNA libraries. Three overlapping clones were assembled and sequenced. Sequence analysis revealed an open reading frame which encodes an 800-amino acid polypeptide (Fig. 1A) with a predicted mass of 89.0 kDa. This open reading frame contains all three of the peptide sequences derived from the natural hTAFII100 (Fig. 1A). When a rapid amplification of cDNA ends method was employed to obtain a longer 5' cDNA sequence, all transcripts stopped at an identical position 14 nucleotides ahead of the first ATG (data not shown).


Fig. 1. Sequence analysis of human TAFII100 clone. A, the deduced amino acid sequence of hTAFII100. The amino acid sequences obtained by microsequencing of hTAFII100-derived peptides are underlined (dashed lines). Five amino acid residues which differ from the sequence reported by Dubrovskaya et al. (12) are indicated by asterisks. Arrows indicate the WD-40 repeat sequences. B, sequence alignment of the WD-40 repeats of the human TAFII100, Drosophila TAFII80, and yeast TAFII90, based on the consensus sequence reported by Neer et al. (26).
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To verify that the polypeptide encoded by the cloned cDNA is an intrinsic human TFIID subunit, antibodies were generated with bacterially expressed N-terminal (1-455) and C-terminal (456-750) portions of this protein. Western blot analysis with antigen affinity-purified antibodies (Fig. 2A) confirmed the presence of a 100-kDa immunoreactive band in both HeLa nuclear extract (lane 1) and immunopurified TFIID (lane 2). Furthermore, the cDNA was subcloned into pBluescript vector (Stratagene) and subjected to in vitro transcription and translation in the presence of [35S]methionine. The size of the 35S-labeled protein (Fig. 2B, lane 2) was indistinguishable from that of the 100-kDa subunit of TFIID detected by Western blot (Fig. 2B, lane 1). A control, antisense construct does not produce this 100-kDa protein (data not shown). When a cDNA encoding the corresponding FLAG-tagged protein (with an extra 18 amino acids) was expressed in Sf9 cells, a correspondingly larger protein was detected both by the purified anti-hTAFII100 antibody (Fig. 2A, lane 3) and by anti-FLAG antibody (data not shown). Thus, although the longest cDNA does not contain a stop codon preceding the open reading frame, these data indicate that it encodes a full-length human TAFII100. This conclusion is further supported by comparison with a rat TAFII100 cDNA, which encodes a protein whose N-terminal region is nearly identical with that of human TAFII100.3 The hTAFII100 subunit in anti-TBP antibody-purified human TFIID (Ref. 17 and this paper) is equivalent to the hTAFII95 subunit in anti-FLAG antibody-purified human fTFIID (22), which hereafter will be designated hTAFII100 in accordance with more general convention (12, 17).


Fig. 2. Identification of the cDNA-encoded protein as the 100-kDa human TFIID subunit. A, affinity-purified anti-hTAFII100(1-455) antibody was used to detect hTAFII100 in HeLa nuclear extract (lane 1), in a natural TFIID complex (lane 2), and in baculovirus-expressed FLAG-hTAFII100 (lane 3). The TFIID complex was affinity-purified from HeLa nuclear extracts on an anti-hTAFII100 antibody column and, as shown in Fig. 3D, contains TBP and all other TAFs. B, the plasmid containing the cDNA encoding hTAFII100 was used to produce in vitro translated and [35S]methionine-labeled hTAFII100. The proteins present in 1 µl of HeLa nuclear extract (lane 1) and 2 µl of [35S]methionine-labeled reticulocyte extract (lane 2) were resolved by 6% SDS-PAGE and transferred onto a nitrocellulose membrane. Lane 1 was immunoblotted with anti-hTAFII100(456-750) antibody and lane 2 was subjected to autoradiography. Arrows indicate the specific 100-kDa protein band. The fast moving products in lane 2 reflect degraded or premature-termination products of hTAFII100, except for one band (marked by asterisk) that also appeared in the control reticulocyte lysate.
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A GenBank search indicated that hTAFII100 shares highly conserved amino acid sequences with Drosophila TAFII80 (dTAFII80) and yeast TAFII90 (yTAFII90), particularly in the C-terminal regions of these proteins (25-27). Fig. 1B shows an alignment of hTAFII100, dTAFII80, and yTAFII90 according to the highly conserved WD-40 repeat consensus sequences (28) that were previously reported in dTAFII80 (25, 26) and yTAFII90 (27). Recent data from the crystal structure of a G protein beta gamma dimer (29, 30) indicate that the WD-40 repeats form a compact beta -propeller structure, leading to speculation that the C terminus of hTAFII100 (as well as that of dTAFII80 and yTAFII90) may form a similar compact structure.

While this manuscript was in preparation, Dubrovskaya et al. (12) also reported the molecular cloning of human TAFII100. Compared to their sequence, the hTAFII100 cDNA sequence reported here contains several nucleotide changes. Our sequence encodes an alanine rather than a threonine at position 126, a phenylalanine rather than a leucine at position 300, a proline rather than a serine at position 455, an alanine rather than a valine at position 469, and an additional threonine at position 778. Currently we do not know the reason for these differences, although the phenylalanine at position 300 is conserved in yTAFII90 and the additional threonine at position 778 provides better sequence alignment of hTAFII100 with both dTAFII80 and yTAFII90 (containing serine and threonine, respectively, at this position).

hTAFII100 Is Associated with Transcriptionally Active Forms of TFIID Containing Other TAFs

Since hTAFII100 has not yet been implicated in any TFIID function, and to verify whether it is in fact in an active TFIID complex, we next determined whether affinity-purified antibodies raised against the N- and C-terminal regions of hTAFII100 can inhibit transcription in vitro. Initially, using strong TATA-containing promoters, we failed to observe inhibition of transcription by direct addition of purified antibodies to reactions containing either nuclear extract or highly purified RNA polymerase II and general initiation factors (data not shown, see also below). We then used affinity-purified antibodies to deplete TFIID from nuclear extracts by cycling the extracts through an antibody column. A Western blot analysis of the treated extracts revealed that more than 90% of the various type II TAFs (including hTAFII100) were depleted by antibody specific for the N-terminal portion of hTAFII100, whereas none were depleted by a control antibody column (Fig. 3A, lane 3 versus lane 1 and data not shown). In contrast, less than 50% of the total TBP was removed (Fig. 3A), consistent with the presence of TBP in complexes utilized for transcription by RNA polymerases I and III (31). Moreover, immunoprecipitates obtained with the N-terminal specific antibody were shown to contain TBP and all type II TAFs (Fig. 3D, and data not shown).

When tested in a transcription assay with templates containing strong core promoters (Fig. 3B), the nuclear extract immunodepleted with the N-terminal-specific antibody was deficient in both basal (lower arrow) and activator (GAL4-VP16)-dependent (upper arrow) transcription (lanes 7-9) relative to the control extract (lanes 1-3). When purified TFIID was added back to the hTAFII100-depleted nuclear extract, both transcription activities (basal and activator-dependent) were restored (Fig. 3C, lanes 3-8 versus lanes 1-2). In contrast, antibody specific for the C-terminal portion of hTAFII100 failed to deplete TFIID subunits from nuclear extract (Fig. 3A, lane 2 versus lane 1) and had no effect on either the basal or the activator-dependent functions of TFIID (Fig. 3B, lanes 4-6 versus lanes 1-3). Since the antibodies to the N- and C-terminal portion of hTAFII100 show comparable reactions with hTAFII100 in immunoblots (Fig. 2), this could indicate that the C-terminal portion of hTAFII100 (containing the WD-40 repeats) is buried inside the TFIID complex. Taken together, these data indicate that hTAFII100 is an integral (tightly associated) component of essentially all transcriptionally active forms of TFIID, and that at least part of the N-terminal half of hTAFII100 is exposed on the surface of the TFIID complex and potentially capable of interacting with other factors.

hTAFII100 Interactions with Other Subunits of TFIID

We next investigated possible interactions of hTAFII100 with TBP and other TAFs both in vivo and in vitro. To examine intracellular (in vivo) interactions, individual TFIID subunits were expressed either alone or together with a FLAG epitope-tagged hTAFII100 (FLAG-hTAFII100) in Sf9 cells. Derived cell lysates were immunoprecipitated either with anti-FLAG antibodies (for hTAFII80, hTAFII31, hTAFII20, and TBP) or with anti-hTAFII100 antibodies (for HA-hTAFII250, FLAG-hTAFII55, and FLAG-hTAFII28). As revealed by immunoblot analysis, comparable levels of other TFIID subunits were expressed in the absence (Fig. 4A, lane 1) and presence (lane 3) of co-expressed hTAFII100. When hTAFII100 was co-expressed, anti-hTAFII100 antibodies co-immunoprecipitated significant levels of TBP, hTAFII31, hTAFII80, and hTAFII250, lower but readily detectable levels of hTAFII28 and hTAFII20, and no detectable hTAFII55 (Fig. 4A, lane 4). In control assays, with extracts from cells lacking hTAFII100, no significant amounts of other TFIID subunits were co-precipitated (Fig. 4A, lane 2). In these analyses, recombinant virus inputs were adjusted so that hTAFII100 (when expressed) was limiting (expressed at a cellular level comparable to that of HeLa cells), while the co-expressed TFIID subunits were expressed at very high levels (over 10-50-fold greater). Hence, it was expected, even for strong interactions, that only a small fraction of the co-expressed interacting TFIID subunits would be co-immunoprecipitated with hTAFII100.

To test whether the indicated hTAFII100 interactions are direct, in vitro binding assays were performed with highly purified recombinant TBP and hTAFs. In these assays roughly equivalent amounts of recombinant proteins were incubated prior to immunoprecipitation. As shown in Fig. 4B, significant amounts of TBP, hTAFII80, hTAFII28, and hTAFII31, but no hTAFII250, TAFII55, or hTAFII20, were detected in the anti-hTAFII100 immunoprecipitates. These data indicate that hTAFII100 can interact directly with TBP, hTAFII80, hTAFII31, and hTAFII28 (see also below) and are consistent with the intracellular interaction data described above. The failure to see significant interactions of hTAFII100 with hTAFII250 and hTAFII20, as well as the apparently weak interaction between hTAFII100 and hTAFII31, contrasts with the results of the intracellular interaction assays. Although this could reflect indirect interactions of these proteins in the intracellular assays, it could also reflect limiting concentrations of properly folded TAFs or interference by the anti-hTAFII100 antibodies used in the in vitro interaction assays. We have therefore utilized two alternative assays to discern direct interactions.

The first alternative assay analyzed direct binding of purified in vitro translated hTAFII100 (35S-labeled) to purified hTAFII31, purified hTAFII80, and a purified in vivo assembled complex of hTAFII31 and hTAFII80. The purity of these latter components is shown in Fig. 4C. In the interaction assay (Fig. 4D) hTAFII100 showed strong hTAFII31 (lane 3) and hTAFII80 (lane 4) interactions, which paralleled those observed in the intracellular assays, as well as interactions with the stable preformed hTAFII31-hTAFII80 complex (lane 5). The second assay involved reconstitution of purified recombinant proteins and direct visualization by SDS-PAGE and silver staining, and revealed the formation of a stable TBP·hTAFII100·hTAFII250 complex whose composition is shown in Fig. 4E. This result is consistent with the previously documented in vivo interactions of hTAFII100 with TBP and hTAFII250.

Except for the case of hTAFII55, our present observations on hTAFII100 interactions are in agreement with those reported by Dubrovskaya et al. (12). However, we report novel direct interactions of hTAFII100 with the histone H4-like hTAFII80, the histone H3-like hTAFII31, and the naturally occurring complex of these two TAFs. Interactions with hTAFII80 and hTAFII31 were not examined by Dubrovskaya et al. (12) and appear in our analyses to represent the strongest hTAFII100 interactions.

Anti-hTAFII100 Antibodies Selectively Inhibit TATA-less Core Promoter Function

Although not essential for basal transcription from conventional TATA-containing promoters, TAFs are absolutely required, along with other cofactors, for basal transcription from certain initiator-containing promoters that lack TATA elements (23, 24). Specific TAF requirements for these functions have not been described. However, a position-specific photocross-linking study has localized hTAFII100 near positions -9/-8 in a TFIID-Ad2ML promoter complex (5), leading to speculation that hTAFII100 might play a role in core promoter recognition at or near initiator (Inr) elements. To explore this possibility, as well as possible promoter-specific functions, we compared the effects of affinity-purified anti-hTAFII100(1-455) antibody on basal and activated transcription from strong TATA-containing promoters (pG5HMC2AT and pMLDelta 53, also employed in the analysis of Fig. 3) and on basal transcription from the TATA-less Inr-containing TdT promoter. Because basal transcription from the TdT core promoter is much weaker (about 50-fold lower) than that from the TATA-containing AdML promoter, an Inr-less TATA-containing promoter (human Hsp70) of comparable strength was used as a control. As shown in Fig. 5A, high concentrations of anti-hTAFII100(1-455) antibody showed only marginal inhibitory effects on basal and GAL4-VP16-activated transcription from the pMLDelta 53 and pG5HMC2AT templates. In contrast, similar amounts of anti-hTAFII100(1-455) antibody dramatically decreased transcription from the TATA-less Inr-containing TdT template, while showing no significant effect on the Inr-less TATA-containing Hsp70 promoter (Fig. 5, lanes 1-4). As further evidence of specificity, comparable amounts of a control anti-HA antibody had no effect on transcription from either the TdT or the Hsp70 template (Fig. 5B, lanes 5-7). Altogether, these results raise the interesting possibility that hTAFII100 may be involved in core promoter-specific effects, possibly relating to initiator function.


DISCUSSION

The TAF subunits of TFIID have been implicated as targets for both gene-specific activators (1, 3) and global repression mechanisms during mitosis (8), and as modulators (negative and positive) of TFIID binding to diverse core promoter elements (2, 3). Although not generally essential for basal transcription directed by TATA elements in core promoters, TAFs are essential for the function of other core promoter elements (initiator, downstream promoter element) either alone or in conjunction with TATA elements (reviewed in Ref. 2). Related to these functions, a number of studies have described interactions of TFIID subunits with each other (1, 9-16), with specific activators and general initiation factors (1-3), and with specific core promoter sequences (1-3, 32, 33), in addition to regulatory (post-translational) modifications of TFIID subunits (8). To investigate these questions, as part of our ongoing efforts to understand the assembly, structure, and function of human TFIID, we report here the cloning of a cognate cDNA and initial characterization of the 100-kDa subunit (hTAFII100) of human TFIID.

The cloned cDNA-encoded polypeptide described here appears to represent the natural hTAFII100 on the basis of amino acid sequences, size, and immunological cross-reactivity. That it is a bona fide TAF is suggested by sequence relationships to Drosophila (dTAFII80, Refs. 25 and 26) and yeast (yTAFII90, Ref. 27) TAFs and by functional tests. Of particular significance is the demonstration that hTAFII100 is a component of all transcriptionally active forms of TFIID in nuclear extracts. To date, however, no specific function of TFIID in basal or activator-enhanced transcription has been reported.

One of the striking features of hTAFII100 and its Drosophila and yeast homologues is the presence of perfect WD-40 repeats (6 in hTAFII100) in the C-terminal half of the molecule. The WD-40 repeats were originally found in the beta -subunit of the G protein (34), a heterotrimeric complex that transduces signals from transmembrane receptors to other second messenger-generating effectors, but is now evident in a various of regulatory proteins (28). A role for the WD-40 repeats in facilitating protein-protein interaction is evident from mutational studies of yeast transcription factors (35), and from structural studies showing that WD-40 repeats in the G protein beta  subunit form a compact beta -propeller structure and interact directly with the G protein gamma  subunit (29, 30). Although it was shown that WD-40 repeats are dispensable for dTAFII80 interactions with two Drosophila TFIID subunits (TBP and dTAFII110, Ref. 24) and for co-immunoprecipitation of hTAFII100 with TFIID (12), some of our observations are consistent with the possibility of hTAFII100 interactions with other TAFs through the WD40 repeats. First, unlike antibodies directed against the N-terminal half of hTAFII100, antibodies directed toward the C-terminal portion containing the WD-40 repeats do not immunoprecipitate TFIID. Second, the C-terminal half of hTAFII100 shows direct interactions with other TFIID subunits.4 Hence, it is likely that at least part of the N-terminal half of hTAFII100 is on the surface of TFIID while the C-terminal WD-40 repeat regions may, at least in part, be buried within TFIID. Although Deubrovskava et al. (12) reported that a monoclonal antibody recognizing a C-terminal epitope could interact with TFIID and modestly reduce basal transcription (and RAP30 interactions), this epitope is outside the WD-40 repeat region.

While there have been no reports of hTAFII100 interactions with any activators, in contrast to what has been observed for many other TAFs (1, 3), in vivo and in vitro protein-protein interaction assays have suggested interactions that are potentially important for TFIID assembly and stability. In agreement with the recent report of Dubrovskaya et al. (12), we have detected physical interactions of hTAFII100 with TBP, hTAFII250, hTAFII28, and hTAFII20. However, we also have observed novel and considerably stronger interactions of hTAFII100 with hTAFII80 and hTAFII31, and with a complex of these proteins. Of significance in this regard are previous observations that hTAFII80 and hTAFII31 (and their Drosophila counterparts) have regions that are homologous to histones H4 and H3, respectively, and form a stable structure similar to the H3-H4 heterotetramer in the nucleosome (15, 16). In addition, hTAFII20 is related in sequence to histone H2B, shows interactions with hTAFII80 that parallel the histone H2B-H4 interactions, forms a dimer which mirrors the H2B-H2A interactions, and is present in a molar ratio of about four in TFIID (13, 15).

These results have led to the proposal that these histone-like TAFs form a nucleosomal-like "octamer" within TFIID during assembly or function (15, 16). Our present results, showing strong interactions of hTAFII100 with hTAFII80 and hTAFII31, and modest interactions with hTAFII20, raise the possibility that hTAFII100 may help stabilize interactions of the histone-like TAFs, perhaps within an octamer structure. This notion is particularly appealing, since it has proved difficult to isolate a stable complex of all three histone-like TAFs in the expected stoichiometry5 and since it is known that the core histone octamer structure is unstable at physiological salt concentrations in the absence of DNA (36). Moreover, recent studies in yeast have identified mutated forms of yTAFII90 (the yeast homologue of hTAFII100) as suppressors of a mutant form of yTAFII60 (the yeast homologue of hTAFII80),5 providing strong evidence for in vivo interactions between these TAFs. Also consistent with more stable interactions of hTAFII100 with hTAFII80 and hTAFII31 than with other TAFs, systematic TFIID disruption studies with anti-TBP- and anti-hTAFII20-immobilized TFIID showed concomitant dissociation of hTAFII100, hTAFII80, hTAFII31, and hTAFII250 (13). Similarly, TFIID assembly studies have indicated that hTAFII100 binds more strongly to an hTAFII80·hTAFII31 complex relative to an hTAFII250·TBP complex (Fig. 4 and data not shown). Other studies have shown that hTAFII100, but not the histone-like TAFs (with the possible exception of hTAFII31), are efficiently cross-linked to a region just upstream of the transcription initiation site in a TFIID-Ad2ML promoter complex (5). This leaves open the possibility that the histone-like TAFs are positioned for previously described interactions (some activator induced) of TFIID with downstream sequences (reviewed in Refs. 2 and 5). Alternatively, they may be buried within the TFIID complex and play an integral role in its topological organization and function.

Another novel feature of the current study is the finding that anti-hTAFII100 antibodies selectively inhibit the function of an Inr-containing TATA-less promoter (TdT), when compared to a TATA-containing promoter of comparable strength (Hsp70). These results are consistent with the unique TAF requirement for basal transcription by Inr-containing TATA-less promoters (23, 24) and with the above-mentioned localization of hTAFII100 near the initiation site when TFIID is bound to the TATA- and Inr-containing AdML promoter (5). These observations raise the interesting possibility that hTAFII100 may have core promoter-specific functions, being relatively more important for the function of Inr-containing TATA-less promoters. However, present uncertainty about the factors, whether TAFs or other essential cofactors (2, 24), that directly contact such promoters limits consideration of possible mechanisms for hTAFII100 function (for example, whether it recognizes DNA directly, acts as a bridging factor to an Inr-bound protein, or is critical for a certain TFIID conformation). Nonetheless, our studies indicate a fundamental difference in the topology and/or function of the corresponding TFIID-promoter complex that makes it uniquely sensitive to the presence of anti-hTAFII100 antibody. Further studies with reconstituted TFIID specifically lacking hTAFII100 or containing mutated forms of hTAFII100 should answer these questions.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants CA42567 and AI37327 (to R. G. R) and GM45258 (to M. H.), Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture in Japan (to M. H.), fellowships from the Charles H. Revson/Norman and Rosita Winston Foundation (to E. M.), the Deutsche Forschungsgemeinschaft (to T. O.), and the Uehara Memorial Foundation (to R. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U80191[GenBank].


   Present address: Center for Molecular and Developmental Biology, Faculty of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-01, Japan.
par    To whom correspondence should be addressed. Tel.: 212-327-7600; Fax: 212-327-7949; E-mail: roeder{at}rockvax.rockefeller.edu.
1   The abbreviations used are: TBP, TATA box-binding protein; TF, transcription factor; TAF, TBP-associated factor; hTAF, human TAF; dTAF, Drosophila TAF; yTAF, yeast TAF; HA, hemagglutinin; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; TdT, terminal deoxynucleotidyltransferase.
2   M. Guermah and R. G. Roeder, unpublished data.
3   T. Yamamoto and M. Horikoshi, unpublished data.
4   Y. Tao and R. G. Roeder, unpublished data.
5   Y. Nakatani, personal communication.

Acknowledgments

We are grateful to C.-M. Chiang for anti-hTAFII55 antibody, A. Hoffmann for bacterial expression vectors (6HisT-pRSET and pGEX-2TL(+)) and anti-hTAFII20 antibody, S. Stevens for anti-hTAFII135 antibody, and X. L. Shi for recombinant GAL4-VP16 protein. We also thank other members of the Roeder laboratory, especially H. Xiao and Z. X. Wang, for helpful suggestions, and Y. Nakatani (National Institutes of Health) for communicating results prior to publication. Peptide sequencing was performed by the Protein Sequencing Facility of The Rockefeller University.


Note Added in Proof

Tanese et al. (Tanese, N., Saluja, D., Vassallo, M. F., Chen, J.-L., and Admon, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13611-13616) have recently reported the cloning of a cDNA encoding hTAFII100, and their deduced protein sequence matches exactly that reported here.


REFERENCES

  1. Burley, S. K., and Roeder, R. G. (1996) Annu. Rev. Biochem. 65, 769-799 [CrossRef][Medline] [Order article via Infotrieve]
  2. Roeder, R. G. (1996) Trends Biochem. Sci. 21, 327-335 [CrossRef][Medline] [Order article via Infotrieve]
  3. Verrijzer, C. P., and Tjian, R. (1996) Trends Biochem. Sci. 21, 338-342 [CrossRef][Medline] [Order article via Infotrieve]
  4. Kaiser, K., and Meisterernst, M. (1996) Trends Biochem. Sci. 21, 342-345 [CrossRef][Medline] [Order article via Infotrieve]
  5. Oelgeschläger, T., Chiang, C. M., and Roeder, R. G. (1996) Nature 382, 735-738 [CrossRef][Medline] [Order article via Infotrieve]
  6. Walker, S. S., Reese, J. C., Apone, L. M., and Green, R. (1996) Nature 383, 185-188 [CrossRef][Medline] [Order article via Infotrieve]
  7. Moqtaderi, Z., Bai, Y., Poon, D., Weil, P. A., and Struhl, K. (1996) Nature 383, 188-191 [CrossRef][Medline] [Order article via Infotrieve]
  8. Segil, N., Guermah, M., Hoffmann, A., Roeder, R. G., and Heintz, N. (1996) Genes Dev. 10, 2389-2400 [Abstract]
  9. Weinzier, R. O. J., Ruppert, S., Dynlacht, B. D., Tanese, N., and Tjian, R. (1993) EMBO J. 12, 5303-5309 [Abstract]
  10. Kokubo, T., Gong, D. W., Wootton, J. C., Horikoshi, M., Roeder, R. G., and Nakatani, Y. (1994) Nature 367, 484-487 [CrossRef][Medline] [Order article via Infotrieve]
  11. Mengus, G., May, M., Jacq, X., Staub, A., Tora, L., Chambon, P., and Davidson, I. (1995) EMBO J. 14, 1520-1531 [Abstract]
  12. Dubrovskaya, V., Lavigne, A-C., Davidson, I., Acker, L., Staub, A., and Tora, L. (1996) EMBO J. 15, 3702-3712 [Medline] [Order article via Infotrieve]
  13. Hoffmann, A., and Roeder, R. G. (1996) J. Biol. Chem. 271, 18194-18202 [Abstract/Free Full Text]
  14. Hisatake, K., Ohta, T., Takada, R., Guermah, M., Horikoshi, M., Nakatani, Y., and Roeder, R. G. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8195-8199 [Abstract]
  15. Hoffmann, A., Chiang, C. M., Oelgeschläger, T., Xie, X., Burley, S. K., Nakatani, Y., and Roeder, R. G. (1996) Nature 380, 356-359 [CrossRef][Medline] [Order article via Infotrieve]
  16. Xie, X., Kokubo, T., Cohen, S. L., Mirza, U. A., Hoffmann, A., Chait, B. T., Roeder, R. G., Nakatani, Y., and Burley, S. K. (1996) Nature 380, 316-322 [CrossRef][Medline] [Order article via Infotrieve]
  17. Takada, R., Nakatani, Y., Hoffmann, A., Kokubo, T., Hasegawa, S., Roeder, R. G., and Horikoshi, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11809-11813 [Abstract]
  18. Fernandez, J., Andrews, L., and Mische, S. M. (1994) Anal. Biochem. 218, 112-118 [CrossRef][Medline] [Order article via Infotrieve]
  19. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  20. Chiang, C.-M., and Roeder, R. G. (1995) Science 267, 531-536 [Medline] [Order article via Infotrieve]
  21. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  22. Chiang, C.-M., Ge, H., Wang, Z., Hoffmann, A., and Roeder, R. G. (1993) EMBO J. 12, 2749-2762 [Abstract]
  23. Martinez, E., Chiang, C.-M., Ge, H., and Roeder, R. G. (1994) EMBO J. 13, 3115-3126 [Abstract]
  24. Martinez, E., Zhou, Q., L'Etoile, N. D., Oelgeschläger, T., Berk, A. J., and Roeder, R. G. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11864-11868 [Abstract]
  25. Dynlacht, B. D., Weinzier, R. O., Admon, A., and Tjian, R. (1993) Nature 363, 176-179 [CrossRef][Medline] [Order article via Infotrieve]
  26. Kokubo, T., Gong, D. W., Yamashita, S., Takada, R., Roeder, R. G., Horikoshi, M., and Nakatani, Y. (1993) Mol. Cell. Biol. 13, 7859-7863 [Abstract]
  27. Reese, J. C., Apone, L., Walker, S. S., Griffin, L. A., and Green, M. R. (1994) Nature 371, 523-527 [CrossRef][Medline] [Order article via Infotrieve]
  28. Neer, E. J., Schmidt, C. J., Nambudripad, R., and Smith, T. F. (1994) Nature 371, 297-300 [CrossRef][Medline] [Order article via Infotrieve]
  29. Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058 [Medline] [Order article via Infotrieve]
  30. Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319 [CrossRef][Medline] [Order article via Infotrieve]
  31. Hernandez, N. (1993) Genes Dev. 7, 1291-1308 [CrossRef][Medline] [Order article via Infotrieve]
  32. Verrijzer, C. P., Yokomori, K., Chen, J.-L., and Tjian, R. (1994) Science 264, 933-941 [Medline] [Order article via Infotrieve]
  33. Burke, T. W., and Kadonaga, J. T. (1996) Genes Dev. 10, 711-724 [Abstract]
  34. Fong, H. K. W., Hurley, J. B., Hopkins, R. S., Miake-Lye, R., Johnson, M. S., Doolittle, R. F., and Simon, M. I. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2162-2166 [Abstract]
  35. Komachi, K., Redd, M. J., and Johnson, A. D. (1994) Genes Dev. 8, 2857-2867 [Abstract]
  36. Eickbush, T. H., and Moudrianakis, E. N. (1978) Biochemistry 17, 4955-4964 [Medline] [Order article via Infotrieve]

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