cDNA clone encoding Drosophila transcription factor TFIID.

Abstract

Proper initiation of transcription by RNA polymerase II requires the TATA-consensus-binding transcription factor TFIID. A cDNA clone encoding the Drosophila TFIID protein has been isolated and characterized. The deduced amino acid sequence reveals an open reading frame of 353 residues. The carboxyl-terminal 180 amino acids are approximately 80% identical to yeast TFID and 88% identical to human TFIIID. The amino-terminal portions of the yeast and Drosophila TFIID proteins lack appreciable homology, whereas the Drosophila and human amino termini appear qualitatively similar. In addition, the amino-terminal region of the Drosophila TFID contains several sequence motifs that are found in other Drosophia proteins which appear to regulate transcription. (6)(7)(8). One key general transcription factor is TFIID, which binds to the TATA element in Drosophila (9), human (10, 11), and yeast (12)(13)(14). TFIID has been shown to be required for preinitiation complex formation, suggesting that it functions at an early critical step in the initiation process (15, 16). The yeast and human TFIID proteins function interchangeably to support a basal level oftranscription in vitro, implying significant structural conservation between these molecules (12)(13)(14).The recent cloning ofthe yeast (Saccharomyces cerevisiae) TFIID gene has allowed an examination of the functional and putative structural properties of this transcription factor (17-21 A Drosophila melanogaster embryonic (0-24 hr) cDNA library in AZAP II (Stratagene) was screened by hybridization with a cloned PCR probe. Hybridizations were performed at 400C in 6x SSPE/0.25% nonfat dried milk/50%o deionized formamide. Filters were washed at 650C in 2x SSC (lx = 0.15 M NaCl/15 mM sodium citrate, pH 7.0)/0.1% NaDodSO4. After plaque purification, individual cDNA inserts were recovered in the form of chimeric pBluescript SK(+) phagemids by in vivo excision from the A vector as described by the library supplier.Overproduction of Recombinant Drosophila TFVD. The conserved region ofDrosophila TFIID was subcloned into an Escherichia coli expression system as follows. The Apa I-EcoRI fragment of the Drosophila TFIID cDNA insert encoding the carboxyl-terminal 1% amino acid residues was inserted into the pET-8c (T7 expression) vector (27) at the Nco I-BamHI restriction sites. A BamHI site was added to the EcoRI site of the TFIID insert by ligation of an EcoRIBamHI (duplex oligonucleotide) adaptor (New England Biolabs). The modified TFIID insert was then ligated into pET-8c cut with BamHI and Nco I. The 3' overhang of the TFIID Apa I site was joined to the 5' overhang of the Nco I site of pET-8c by use of an eight-base "bridging oligonucleotide" with sequence complementarity to both the Nco I and Apa I overhangs (5'-CATGGGCC-3'). The trimolecular ligation joined the initiating methionine codon of the pET-8c vector in-frame to the Gly-158 codon of the TFIID open reading frame. Plasmid DNA samples prepared from XL1-Abbreviations: PCR, polymerase chain reaction; IPTG, isopropy...

Promoter melting and TFIID complexes on Drosophila genes in vivo.

“…4, has no TATA box). We reasoned that this protection is most likely the result of the binding of the TBP component of TFIID to the TATA box (Parker and Topol 1984;Wu 1984;Hoey et al 1990;Muhich et al 1990}. The protection of these T residues is the same in heat-shocked and non-heat-shocked cells, a finding consistent with earlier chromatin footprinting experiments performed in isolated nuclei where a constitutive protection of the TATA region from exonuclease III and TBP protects the TATA box from reacting with KNInO 4 are presented in the Discussion.…”

TBP, a universal eukaryotic transcription factor?

“…In the human and Drosophila proteins, glutamine stretches are marked {boxes labeled Q). References: Human {Hoffmann et al 1990b; Kao et al 1990;Peterson et al 19901; mouse (Tamura et al 1991};Drosophila {Hoey et al 1990;Muhich et al 1990);A. tbaliana {Gasch et al 1990};maize (Haass and Feix 1992};potato {Holdsworth et al 1992};wheat-1 {Kawata et al 1992};wheat-2 {Apsit et al 1993); ScbJzosaccbaromyces pom be (Fikes et al 1990;Hoffmann et al 1990a);S.…”

“…This was a major breakthrough because, unlike mammalian TFIID, the yeast activity could be purified to homogeneity and turned out to correspond to a single polypetide of 27 kD (Cavallini et al 1989;Hahn et al 1989;Horikoshi et al 1989a;Schmidt et al 1989). The cDNA encoding the yeast TATA box-binding protein (TBP) was then cloned by several groups (Cavallini et al 1989;Hahn et al 1989;Horikoshi et al 1989b;Schmidt et al 1989), and the protein sequence information was used to isolate TBP-encoding 11742 cDNAs from several other species, including Drosophila (Hoey et al 1990;Muhich et al 1990) and humans (Hoffman et al 1990b;Kao et al 1990;Peterson et al 1990). …”

Drosophila 230-kD TFIID subunit, a functional homolog of the human cell cycle gene product, negatively regulates DNA binding of the TATA box-binding subunit of TFIID.

“…Briefly, the blot was denatured with 6 m guanidine hydrochloride in buffer A [9,0 mM Tris-HC1 (pH 7.9), 0.9,5 M KC1, 0.2 mM EDTA, 10 m~ 2-mercaptoethanol, 0.5 mM PMSF, 10% {vol/vol) glycerol], followed by successive treatments with 3.0, 1.5, 0.75, and 0.375 M guanidine hydrochloride in buffer A. After blocking, the renatured blot was incubated with 500 ~1 of buffer A containing 1% skim milk mixed with 50 ~1 of rabbit reticulocyte lysate containing ass-labeled TFIID~ for 12 hr at 4~ RNA synthesis and aSS-labeled protein synthesis from Drosophila (Hoey et al 1990;Muhich et al 1990), human (Hoffmann et al 1990), and yeast (Horikoshi et al 1989b) TFIID~ cDNAs with rabbit reticulocyte lysate were performed according to a standard protocol (Promega). The blot was then washed with buffer A, dried, and exposed at room temperature.…”

“…The purified protein was then injected into New Zealand white rabbits to prepare specific antiserum. The antisera for TFIID~ was produced by immunizing the synthetic peptide corresponding to amino acids position 30-47 (Hoey et al 1990;Muhich et al 1990) and affinity-purified as described (Kokubo et al 1993).…”