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Proc. Natl. Acad. Sci. USA Vol. 86, pp. 7785-7789, October 1989 Biochemistry Yeast TATA-box factor ( HID/DNA binding /DNA sequence/protein sequence) MARTIN C. SCHMIDT, C. CHENG KAO, Rui PEI, AND ARNOLD J. BERK* Institute and the Department of Microbiology, University of California, Los Angeles, CA 90024-1570 Communicated by Richard E. Dickerson, July 19, 1989 (received for review June 22, 1989)

ABSTRACT The first step in the transcription of most mammalian cells (4, 11, 12). Moreover, initiation complexes protein-encoding in is the binding of a assembled with yeast TFIID respond to transcriptional stim- transcription factor to the TATA-box element. This ulation by the upstream mammalian factor Spl (13). To TATA-box transcription factor was purified from extracts of analyze TFIID in greater detail, we have purified the S. the yeast by using reconstitution ofin cerevisiae TFIID polypeptide and cloned the gene that en- vitro transcription reactions as an assay. The activity copurified codes it. with a protein whose sodium dodecyl sulfate/polyacrylamide gel mobility is 25 kDa. The sequence of the amino-terminal 21 residues of this protein was determined by sequential Edman MATERIALS AND METHODS degradation. A yeast genomic library was screened with mixed Purification of Yeast TFIID. An extract was prepared from oligonucleotides encoding six residues of the protein sequence. 300 g of the S. cerevisiae strain BJ 1991 (MATa, prbl-1122 The yeast TATA-box factor gene was cloned, and DNA se- pep4-3 leu2 trpl ura3-52) by disruption with glass beads. The quencing revealed a 720-base-pair open reading frame encod- yeast TFIID protein was purified by the method of Bura- ing a 27,016-Da protein. The identity of the clone was con- towski et al. (11). The Superose 12 column fractions were firmed by expressing the gene in Escherichia coli and detecting used in this study. TATA-box factor DNA binding and transcriptional activities in Protein Sequencing. The Superose 12 fractions containing extracts of the recombinant E. coli. The TATA-box factor gene yeast TFIID activity were precipitated with 10% trichloro- was mapped to chromosome five of S. cerevisiae. RNA blot acetic acid and resolved by sodium dodecyl sulfate (SDS)/ hybridization and nuclease S1 analysis indicated that the major PAGE (14). The were transferred to a poly(vinyli- TATA-box factor mRNA is 1.3 kilobases, including an unusu- dine difluoride) membrane (Immobilon-P; Millipore) in 10 ally long 5' untranslated region of 188 ± 5 . mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), Homology searches showed a region of distant similarity to the 1 hr at 50 V 0.4 A. The calcium-binding structures of calpains, a structure that has a pH 11/10% (vol/vol) methanol for and conformation similar to the helix-turn-helix motif of DNA membrane was stained with Coomassie blue, and the band of binding proteins. the 25-kDa polypeptide was excised. The protein was se- quenced at the UCLA Protein Microsequencing Facility with Binding oftranscription factor IID (TFIID; also known as DB an Applied Biosystems model 470A protein sequencer and BTF1) to the TATA-box promoter element is the first equipped with on-line HPLC analysis of the phen- step in the transcription of most protein coding genes in ylthiohydantoin-conjugate amino acids. eukaryotes (1-4). TFIID binding initiates a cascade of bind- Cloning and Sequencing the Yeast Gene Encoding TFIIDt. ing events (4) in which RNA polymerase II and several other A library of S. cerevisiae chromosomal DNA partially di- general transcription factors known as TFIIA, B, E, and F gested by Sau3A and inserted in the BamHI site of the phage (5-9) assemble into a multicomponent complex at the pro- A vector EMBL3 (15) was used to isolate the yeast gene for moter site. This multiprotein complex is required for tran- TFIID. By using the amino-terminal sequence of the 25-kDa scription initiation from a minimal promoter containing only protein, the following 32-fold degenerate oligonucleotide a TATA box and initiation site. The developmental specific- (17-mer) was synthesized: 5'-AA(GA)GA(GA)TT(TC)AA- ity of transcription and the rate of transcription initiation can (GA)GA(GA)GC-3', where the degenerate positions are be determined by factors that interact with enhancers and shown in parentheses. The 17-mer was phosphorylated with upstream promoter elements (10). These regulatory factors phage T4 polynucleotide kinase and [y-32P]ATP and was used are thought to function by influencing events at the TATA to screen the genomic library. Filters were washed at 50°C in box-initiation site region, either by enhancing or interfering a solution containing 3 M (CH3)4NCl by a procedure designed with assembly of the multicomponent initiation complex or for degenerate oligonucleotide probes that optimizes hybrid- by affecting initiation and chain elongation by the polymer- ization for the greatest number of base pairs, independent of ase. the G+C content (16). Nine positive phage clones were Until recently, it has been difficult to characterize the isolated after three successive rounds of screening. The DNA molecular events involved in transcription initiation because from each clone was cleaved with restriction and it has been difficult to purify TFIID, the component that analyzed by Southern blot with the 17-mer as a probe. Six of initiates the process. However, Buratowski et al. (11) re- the phage contained an -2.4-kilobase (kb) EcoRI- cently described the extensive purification ofTFIID from the BamHI fiagment that hybridized to the 17-mer. This fragment yeast Saccharomyces cerevisiae. The yeast factor binds to was subcloned into the EcoRI and BamHI sites of pUC18, the TATA-box region from mammalian as well as yeast resulting in a plasmid designated pAB24. Approximately 1 kb promoters (11) and can participate in a functional initiation of the insert in pAB24 was sequenced by a combination of complex with the other general transcription factors from Abbreviation: TFIID, transcription factor IID. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M26403).

7785 Downloaded by guest on October 4, 2021 7786 Biochemistry: Schmidt et al. Proc. Natl. Acad. Sci. USA 86 (1989) both the dideoxy chain-termination (17) and chemical meth- [y-32P]ATP. The 3' end was mapped by using the HindIII- ods (18) as diagrammed in Fig. 2. BamHI fragment of pAB24 labeled at the HindIII site with Expression of Yeast Gene for TFIII) in Escherichia coli. The DNA polymerase I, Klenow fragment, and all four [a- yeast TFIID gene open reading frame was inserted into theE. 32P]dNTPs. Total yeast RNA (100 pg) was hybridized to 30 coli expression vector pAS1 (19). A BamHI restriction site ng of probe DNA in 50 1ul of 80% formamide buffer (23) at was engineered at the start of the open reading frame by using 350C for 16 hr. Nuclease S1 digestion was with 500 units the polymerase chain reaction to amplify a 1.1-kb fragment (BRL) at 250C for 60 min as described (23). with pAB24 as the initial template. The oligonucleotides used in the polymerase chain reactions (25 pmol each) were an M13 sequencing primer (New England Biolabs; no. 1211), RESULTS which hybridizes to the vector sequence of pAB24 down- Yeast TFIID Activity Copurifies with a 25-kDa Protein. The stream of the BamHI site, and a 25-base oligonucleotide TATA-box transcription factor was purified from extracts of (5'-GGATCCTGAGGAACGTTTAAAGGAG-3'), which hy- S. ce-evisiae by the procedure of Buratowski et al. (11), bridizes to pAB24 near the start site oftranslation ofthe yeast whicn involves the four sequential column chromatography TFIID gene. The amplified fragment was cleaved with steps of heparin-agarose, DEAE-sepharose, Mono S, and BamHI and inserted in both orientations into the BamHI site Superose 12. The Superose 12 sizing column effectively of pAS1. The resulting plasmids contain the yeast TFIID separated the yeast TFIID activity from the bulk of the gene open reading frame in the correct orientation (pASY2D) protein in the Mono S fraction (Fig. 1). The TFIID activity relative to the bacteriophage promoter PL or in the opposite was eluted in a region ofthe included volume consistent with orientation (pASD2Y). The TFIID protein expressed in bac- a native molecular weight of 25 kDa, as was reported by teria is identical to the protein isolated from yeast except for Buratowski et al. (11). Fraction 31 was identified as contain- a slight difference at the amino terminus. The amino-terminal by making sequence of TFIID expressed from pASY2D is NH2- ing the peak of transcriptional activity (Fig. 1B) fMet-Asp-Pro, whereas the yeast-encoded sequence is NH2- dilutions ofthe fractions and assaying the equivalent of0.1 PI Met-Ala-Asp. for complementation of transcription of the adeno- An E. coli AcI857 lysogen was transformed with the E1B gene. Analysis of the proteins present in the plasmids pASY2D and pASD2Y, and 50-ml cultures were Superose 12 fractions by SDS/PAGE revealed that a protein grown at 320C to an OD600 of 1. An equal volume of media with a mobility of 25 kDa was coeluted with the transcrip- preheated to 65°C was added, and the cultures were main- tional activity and was most concentrated in fraction 31. A tained at 42°C for 1 hr. Cells were harvested and suspended smaller protein (-15 kDa) was coeluted with activity in this in 2.5 ml of buffer A [50 mM Tris chloride, pH 8/100 mM experiment, but it was not consistently present in other NaCl/1 mM EDTA/1 mM dithiothreitol/1 mM phenylmeth- Superose 12 column fractions containing yeast TFIID activ- ylsulfonyl fluoride/10% (vol/vol) glycerol]. Lysozyme was ity. Based on the coelution of yeast TFIID activity with the added to 1 mg/ml, and cells were incubated at 4°C for 15 min. 25-kDa protein, we decided to clone the gene encoding the The cells were disrupted by sonication, and the lysate was 25-kDa protein. centrifuged at 10,000 x g for 20 min. The supernatant fraction Amino-Terminal Protein Sequence. Superose 12 fractions was decanted and passed through a 5-ml DEAE-cellulose containing yeast TFIID activity isolated from 300 g of yeast (Whatman; DE-52) column equilibrated in buffer A. The were precipitated and resolved with preparative SDS/PAGE. flow-through fraction was collected and used for activity The proteins were transferred to the poly(vinylidene difluo- assays. ride) membrane, and the band representing the 25-kDa pro- TFIID Activity Assays. Transcriptional and DNA cleavage- tein (-5 pg) was subjected to sequential Edman degradation. inhibition (footprinting) activities of the yeast TFIID protein The amino-terminal 21 residues were determined: were assayed as described (13) with the exception that, in the Ala-Asp-Glu-Glu-Xaa-Leu-Lys-Glu-Phe-Lys-Glu-Ala- footprinting reactions, 50 ng ofpoly(dG-dC)poly(dG-dC) per Asn-Lys-Ile-Val-Phe-Asp-Pro-Asn-Thr, where Xaa repre- reaction was added and MgCl2 was omitted. sents an amino acid that could not be identified. Chromosomal Mapping of the Yeast TFIID Gene. S. cere- Cloning and Sequencing the Yeast TFIID Gene. A 32-fold visiae chromosomes, purchased from Bio-Rad, were electro- redundant 17-base oligonucleotide containing all possible phoresed in a 1% agarose gel in a CHEF-DRII pulse field codon choices for a six-amino acid stretch of the amino system (Bio-Rad) at 150 V at 15°C. The switch time was 70 terminus (Lys-Glu-Phe-Lys-Glu-Ala) was used to screen a sec for the first 15 hr and then 120 sec for the last 12 hr. The genomic library of yeast in A phage EMBL 3 (15). After three chromosomal DNA was first nicked with two 15-min incu- successive rounds of screening, nine positive phage clones bations in 0.25 M HCl and then alkaline-denatured, neutral- were isolated. Six of the phage contained a 2.4-kb EcoRI- ized, and transferred to a nylon membrane by the method of BamHI fragment that hybridized with the 17-mer. The 2.4-kb Southern (20). Hybridization was performed at 37°C in 50% fragment was subcloned into pUC18, generating pAB24. formamide/0.90 M NaCl/0.09 M sodium citrate, pH 7/0.1% The 17-mer was labeled with 32P and used to prime dideoxy- polyvinylpyrrolidone/0. 1% bovine serum albumin/0. 1% sequencing reactions of denatured pAB24. The Ficoll/0.1% SDS/0.1 mg of denatured salmon sperm DNA DNA sequence generated from this experiment encoded an per ml. The probe was prepared by nick-translating the open reading frame that began with the amino acids Lys- EcoRI-HindIII fragment ofpAB24 in the presence of all four Ile-Val-Phe-Asp-Pro-Asn-Thr, matching exactly with the [a-32P]dNTPs. amino acid sequence of the 25-kDa protein determined by Analysis of Yeast TFIID mRNA. A culture of S. cerevisiae Edman degradation. The remainder ofthe open reading frame was grown at 30°C in rich medium to an OD6w of --1. The and the immediately adjacent DNA was sequenced by a cells were collected and the RNA was isolated by extraction combination of dideoxynucleotide and chemical sequencing in hot phenol (21). RNA blot (Northern) transfer and hybrid- techniques (Fig. 2). The plasmid pAB24 contained a 720-bp ization were done as described (22). The DNA probe was the open reading frame that encoded a 27,016-Da protein. The 0.7-kb Dra I-HindIII fragment of pAB24 nick-translated in 21-amino acid sequence determined by Edman degradation the presence of all four [a-32P]dNTPs. The yeast TFIID was present in positions 2-22 of the open reading frame. In mRNA 5' terminus was mapped by nuclease S1 analysis (23) the absence of any posttranslational modifications, the se- using as probe the Spe I-EcoRI fragment ofpAB24 labeled at quence predicts a protein with a net positive charge of +9 at the Spe I site with phage T4 polynucleotide kinase and pH 8, which is consistent with the chromatographic behavior Downloaded by guest on October 4, 2021 Biochemistry: Schmidt et al. Proc. Natl. Acad. Sci. USA 86 (1989) 7787 A - qL r- Z 0 - c K cts l 0o o - Q roe a 5- 0 N, CMj C\J jN C\J C\JCU C\JE Cu]r ro O pr) rO3 n fr~r rl 205- 116- _-.

97- --XNO-a-w - go 4w 40 - 66- "_ I= - -~ FIG. 1. Yeast TFIID activity copurifies with a 25-kDa protein. (A) Protein gel of Superose 12 w - pm do 45- t column fractions. Protein samples were resolved by q-4NO SDS/12% PAGE (14) and stained with silver nitrate -4w4f -Oa (24). Lanes: M, molecular mass standards (sizes in kDa are shown on the left); ON, 4 pg ofprotein from 29- As the material loaded on the Superose 12 column; ~~~- -Y II D 20-37, protein from 20 p4 of Superose 12 column fractions indicated above each lane (fraction 31 contained -60 pg of protein per ml). The mobility of the yeast TFIID protein is indicated (YIID) on 14- - the right. (B) Transcription assays of Superose 12 column fractions. Reconstitution of in vitro tran- B D Lu z scription of the adenovirus EJB gene was assayed z 0 by primer extension (13). Each reaction contained 0T z 29 30 31 32 33 34 35 36 37 HeLa protein fractions capable of initiating tran- scription only when a source of the TFIID protein E B is added. The yeast TFIID fractions added were 0.5 pg of protein that was loaded onto the Superose 12 column (lane ONPUT), no TFIID (lane NONE) or 0.1 dul of Superose 12 column fractions as indicated above each lane. The mobility of the primer exten- sion product of E1B RNA is indicated on the right. of the yeast TFIID protein on DEAE and Mono-S columns (data not shown) and predicts an RNA of 1097 ± 15 bases. used in its purification (11). Taking into account the size ofthe poly(A) tail, this is in good The cloned gene hybridized specifically to chromosome agreement with the size of the major RNA detected by five when the individual chromosomes were resolved with Northern blotting. pulsed-field gel electrophoresis and analyzed by Southern blot (data not shown). In addition, Southern blot analysis of S. cerevisiae genomic DNA revealed that only one yeast gene DISCUSSION hybridized to the clone even at low stringency (data not The gene encoding the S. cerevisiae TATA-box transcription shown). Thus, the yeast TFIID gene is present in a single factor has been cloned and sequenced, and the mRNA copy of chromosome five. expressed from the gene has been mapped. Proof that the Expression ofYeast TFUD inE. coli. To confirm that pAB24 cloned gene encodes yeast TFIID came from expressing the contained the gene for the yeast TATA-box factor, the open cloned gene in E. coli and demonstrating that a protein reading frame was placed in both orientations in the E. coli fraction enriched for the expressed polypeptide isolated from expression vector pAS1 (19). extracts were prepared, these bacterial cells contained TATA-box factor transcrip- and the soluble material was passed through a DEAE col- tional and DNA binding activities. umn. When the proteins present in the flow-through fractions The yeast TFIID gene encodes a polypeptide of 240 were analyzed by SDS/PAGE, a 25-kDa protein was de- residues. Amino-terminal sequencing of the yeast protein tected in the fraction from cells containing pASY2D (correct indicated that the encoded amino-terminal methionine is orientation) but not in cells containing pASD2Y (opposition removed, leaving the second encoded residue, alanine, as the orientation; data not shown). TFIID purified from yeast amino-terminal amino acid of the mature protein. The length complemented in vitro transcription reactions and protected of yeast TFIID predicted by the DNA sequence is in good the TATA-box sequence of the adenovirus EJB promoter agreement with the size of the protein estimated by gel (13). Extracts from E. coli transformed with pASY2D also exclusion chromatography (11) and SDS/PAGE (Fig. 1). contained these activities, while the control E. coli extract Consequently, it is unlikely that many residues are processed did not (Fig. 3). From these data, we conclude that the from the carboxyl terminus of the mature protein. plasmid pAB24 contains the gene encoding the yeast TATA- The mRNA expressed from the yeast TFIID gene was 1.3 box factor. kb in length. The minor RNAs of 1.6-2.8 kb were not Yeast TFIID mRNA. The size of the TATA-box factor consistently detected. Nuclease S1 analysis of the 5' ends of mRNA was analyzed by Northern blot analysis. A predom- the RNAs mapped a major 5' end at 188 ± 5 nucleotides inant RNA of 1.3 kb was detected when probed with 32p_ upstream from the AUG translational start site. The 5' labeled DNA from the yeast TFIID gene (Fig. 4A). In untranslated sequence of the yeast TFIID mRNA is unusu- addition, a diffuse mixture of minor species of RNA ranging ally long for S. cerevisiae. Of 131 yeast mRNAs analyzed in in size from 1.6 to 2.8 kb was detected in this experiment, but a literature search in 1987, 70% had leaders in the range from these minor RNAs were not consistently observed. Nuclease 20 to 60 nucleotides (25). The unusually long leader raises the S1 mapping of the 5' end of the message indicated that the possibility of translational control in the expression of yeast major transcriptional initiation site was 188 + 5 bases up- TFIID. However, the yeast TFIID mRNA leader sequence stream from the start site (Fig. 4B). Nuclease S1 does not contain an upstream AUG as in the GCN4 and CPA1 mapping of the 3' end of the RNA showed a major 3' end 235 mRNAs, which have been shown to be subject to transla- ± 10 bases downstream of the HindIII site shown in Fig. 2 tional control (26, 27). The sequence TATAAAA occurs -75 Downloaded by guest on October 4, 2021 7788 Biochemistry: Schmidt et al. Proc. Natl. Acad. Sci. USA 86 (1989)

E G S DD G B X H M A YeID D2Y Y2D I I 0 0 1 1 O- -O ~ -

--. 0- *~~~~~~~~~~~/ At --0-o -4. -4 I ..-4* -.o 100 bp

-270 -260 -250 -240 -230 AG ATCTACATAT AAAACATGGC TTCAAAGGAT TACTAATGAC TTTTTTTACC -220 -210 -200 -190 -180 -170 TTGATAGGTA TTCTTGATGG TAAGAGCAAA CAAGGGACGT GAAAATTACA GTAGTTACTG -160 -150 -140 -130 -120 -110 TTTTTTTTGG ACTATAAGAT CGGGGGAAAG ATAACACATA AGAAATAAAA CGACTACTAG -100 -90 -80 -70 -60 -50 TTAGACTGCT CTGCGGAAGA AGCAAGGAAG TAAAGGCTGC ATTTTATTTT TCTTTTCTAG 2 3 4 5 6 7 8 9 10 11 12 -40 -30 -20 -10 15 TCCAACATAA ACAGGTGTAT CAAGAGAAAC TTTTTTAATT ATG GCC GAT GAG GAA CGT B D2Y Y2D M A D E E R Y_ehD_ 30 45 60 TTA AAG GAG TTT AAA GAG GCA AAC AAG ATA GTG TTT GAT CCA AAT ACC AGA CAA L K E F K E A N K I V F D P N T R Q 75 90 105 120 mm -* 4A-EIB GTA TGG GAA AAC CAG AAT CGA GAT GGT ACA AAA CCA GCA ACT ACT TTC CAG AGT V W E N Q N R D G T K P A T T F S 135 150 165 180 GAA GAG GAC ATA AAA AGA GCT ACC CCA GAA TCT GAA AAA GAC ACC TCC GCC ACA E E D I K R A T P E S E K D T S A T 1 2 3 4 5 6 195 210 225 TCA GGT ATT GTT CCA ACA CTA CAA AAC ATT GTG GCA ACT GTG ACT TTG GGG TGC S G I V P T L N I V A T V T L G C FIG. 3. Activity of the yeast TFIID protein expressed in E. coli. 240 255 270 285 (A) Protein fractions were assayed for the ability to bind to the AGG TTA GAT CTG AAA ACA GTT GCG CTA CAT GCC CGT AAT GCA GAA TAT AAC CCC adenovirus EIB TATA-box sequence using a DNase I footprinting R L D L K T V A L H A R N A E Y N P assay (13). The reactions in lanes 2 and 3 contained 0.03 and 0.06 Pg 300 315 330 of purified yeast TFIID, respectively. Extracts of E. coli cells (2, 4, AAG CGT TTT GCT GCT GTC ATC ATG CGT ATT AGA GAG CCA AAA ACT ACA GCT TTG K R F A A V I M R I R E P K T T A L or 8 ug of protein of DE-52 fractions, respectively) were present in the reactions in lanes and 7 and in lanes 9, 10, and 345 360 375 390 5, 6, (pASD2Y) ATT TTT GCA TCA GGG AAA ATG GTT GTT ACC GGT GCA AAA AGT GAG GAT GAC TCA 11 (pASY2D). The reactions in lanes 1, 4, 8, and 12 were incubated I F A S GK M V V T G A K S E D D S without any added protein, indicated by 0 above each lane. The DNA 405 420 435 450 probe was the EcoRI-Hindll fragment from pAd1B (13) labeled at AAG CTG GCC AGT AGA AAA TAT GCA AGA ATT ATC CAA AAA ATC GGA TTT GCT GCT the EcoRI site by T4 polynucleotide kinase and ['y.32P]ATP. The K L A S R K Y A R I I 0 K I G F A A position of the EJB TATA-box is shown on the right. (B) Protein 465 480 495 fractions were for TATA factor activity (13). AAA TTC ACA GAC TTC AAA ATA CAA AAT ATT GTC GGT TCG TGT GAC GTT AAA TTC assayed transcriptional K F T D F K I N I V G S C D V K F Reactions contained heat-treated HeLa cell nuclear extract lacking TFIID and one of the 0.06 of TFIID 510 525 540 555 activity (3) following: pg yeast CCT ATA CGT CTA GAA GGG TTA GCA TTC AGT CAT GGT ACT TTC TCC TCC TAT GAG (lane 1), 1 or 5 pg of protein of the DE-52 fractions from E. coli P I R L E G L A F S H G T F S S Y E pASD2Y (lanes 3 or 4, respectively), 1 or 5 pg of protein of the DE-52 570 585 600 fractions from E. coli pASY2D (lanes 5 or 6, respectively), or no CCA GAA TTG TTT CCT GGT TTG ATC TAT AGA ATG GTG AAG CCG AAA ATT GTG TTG additional protein (lane 2). P E L F P G L I Y R M V K P K I V L 615 630 645 660 this is to be the TATA-box TTA ATT TTT GTT TCA GGG AAG ATT GTT CTT ACT GGT GCA AAG CAA AGG GAA GAA Therefore, sequence likely L I F V S G K I V L T G A K R E E promoter element of the yeast TATA-box factor gene. 675 690 705 720 Similarity of the yeast TFIID sequence to other proteins in ATT TAC CAA GCT TTT GAA GCT ATA TAC CCT GTG CTA AGT GAA TTT AGA AAA ATG the National Biomedical Research Foundation and I Y 0 A F E A I Y P V L S E F R K M EMBL/ GenBank data bases was the PROFILE method 730 740 750 760 770 780 analyzed by TGATGGGGAA GGAGTAGACG AAAAMAAAAA AAGGTTTTCT ATTTGTTCCA `TTCAAT (28) and FASTA program (29). No strong similarities were 790 800 810 820 830 840 found to structural motifs associated with known DNA TATTAATGGT CCTCAAAGAA ATAAAAGAAA AGGAAGAAGA AGTAATTGTA ATATCAAACG binding proteins, including the zinc-finger (30), helix- 850 860 870 880 885 turn-helix (31), and leucine-zipper (32) structures. However, GTATATTCTT CTTATTCTAT ATTTATATAT CAATG GTTTTTTATA the PROFILE method found a distant similarity between res- FIG. 2. DNA sequence of the yeast TATA-box factor gene. Se- idues 99-177 of yeast TFIID and the putative calcium binding quencing strategy is shown at the top. The regions of the pAB24 insert regions (33) of calpains, which are Ca2+-activated proteases that were sequenced by the chain-termination method are indicated by (e.g., residues 570-649 of human calpain I heavy chain; ref. and those the arrows beginning with closed circles, sequenced by 34). This similarity scores 3.1 standard deviations above the chemical method are indicated by arrows beginning with open circles. mean for all of a based on the Direction ofthe arrows indicates which strand was sequenced. Restric- comparisons profile calpain This is a tion sites indicated are E (EcoRI), S (Spe I), D (Dra I), G (Bgl II), B (Bal sequences to the 10,100 proteins in the data base. I), X (Xba I), H (HinduI), and M (BamHI). The darker line indicates significant score, being greater than that of distantly related the open reading frame. The sequence of the yeast TATA-box factor calcium binding proteins that have similar calcium-binding gene is shown below with the numbering relative to the translational structures. However, it is unlikely that yeast TFIID binds start site (+1). Amino acid sequence is shown in the single-letter code. calcium, since in the alignment of yeast TFIID and calpain, two aspartic and two glutamic residues that are putative bp upstream of the transcription start site, and purified yeast calcium binding ligands are replaced by lysines in yeast TFIID footprints span this sequence (unpublished results). TFIID. The potential significance of the sequence similarity Downloaded by guest on October 4, 2021 Biochemistry: Schmidt et al. Proc. Natl. Acad. Sci. USA 86 (1989) 7789 A B <-< 3. Nakajima, N., Horikoshi, M. & Roeder, R. G. (1988) Mol. Cell. + + 2Z Biol. 8, 4028-4040. CD OUUCD 5E 4. Buratowski, S. P., Hahn, S., Guarente, L. & Sharp, P. A. (1989) Cell 56, 549-561. 4 .-I* -- |10 5. Matsui, T., Segall, J., Weil, P. A. & Roeder, R. G. (1980) J. aNW Biol. Chem. 255, 11992-11996. 6. Samuels, M., Fire, A. & Sharp, P. A. (1982) J. Biol. Chem. 257, 2.8 ~ . 14419-14427. 1.6-2.01 ---7.... * -90 7. Dignam, J. D., Martin, P. L., Shastry, B. S. & Roeder, R. G. (1983) Methods Enzymol. 104, 582-598. I.3- U i 8. Zheng, X.-M., Moncollin, V., Egly, J. M. & Chambon, P. -U-- (1987) Cell 50, 361-368. * -76 9. Flores, O., Maldonado, E., Burton, Z., Greenblatt, J. & -q- .W.. Reinberg, D. J. (1988) J. Biol. Chem. 263, 10812-10816. -. 10. 0 -. Maniatis, T., Goodburn, S. & Fischer, J. A. (1987) Science 236, .W. -67 1237-1245. 4w 11. Buratowski, S., Hahn, S., Sharp, P. A. & Guarente, L. (1988) Nature (London) 334, 37-42. FIG. 4. Yeast TFIID mRNA analyses. (A) Northern blot. Total 12. Cavallini, B., Huet, J., Plassat, J., Sentenac, A., Egly, J. & yeast RNA (5 was resolved on an agarose pg) gel and after Northern Chambon, J. (1988) Nature (London) 334, 77-80. transfer it was hybridized to a fragment of the yeast TFIID gene labeled with 32p. The autoradiograph is shown. The sizes ofthe yeast 13. Schmidt, M. C., Zhou, Q. & Berk, A. J. (1989) Mol. Cell. Biol. TFIID mRNAs, indicated on the left, were determined relative to 9, 3299-3307. RNA size markers (0.24, 1.35, 2.37, 4.40, 7.46, and 9.49 kb; BRL) 14. Laemmli, U. K. (1970) Nature (London) 227, 680-685. detected by ethidium bromide staining prior to Northern transfer. (B) 15. Frischauff, A. M., Lehrach, H., Poustka, A. & Murray, N. Nuclease S1 analysis. Total yeast RNA (100 ug) was hybridized with (1983) J. Mol. Biol. 170, 827-842. a DNA probe containing the 5' end of the yeast TFIID gene. 16. Woods, W. I., Gitshier, J., Lasky, L. A. & Lawn, R. M. (1985) RNA-DNA hybrids were digested with nuclease S1 and resolved on Proc. Natl. Acad. Sci. USA 82, 1585-1588. an 8% polyacrylamide/8 M urea sequencing gel (lane RNA). The 17. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. same DNA probe was also subjected to Maxam and Gilbert sequenc- Acad. Sci. USA 74, 5463-5467. ing reactions, shown in lanes G, G+A, C+T, and C. DNA size 18. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, markers were also run (lane M), and the fragment sizes in bases are 497-559. shown on the left. 19. Rosenberg, M., Ho, Y. & Shatzman, A. (1983) Methods Enzymol. 101, 123-138. to calpains may be that, as Richardson and Richardson (35) 20. Southern, E. (1975) J. Mol. Biol. 98, 503-517. 21. Domdey, H., Apostol, B., Lin, R. J., Newman, A., Brody, E. have noted, the helix-loop-helix structure of these calcium & Abelson, J. (1984) Cell 39, 611-621. binding sites has a similar conformation to the helix- 22. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. USA 77, 5201- turn-helix motif of DNA binding proteins. Mutagenic and 5205. structural studies of yeast TFIID will be required to deter- 23. Berk, A. J. (1989) Methods Enzymol. 180, 334-347. mine if the region of similarity to calpain functions in DNA 24. Wray, W., Boulikas, T., Wray, V. P. & Hancock, R. (1981) binding. Anal. Biochem. 181, 197-203. Isolation of the yeast TFIID gene should facilitate further 25. Cigan, A. M. & Donahue, T. F. (1987) Gene 59, 1-18. studies on the function of this which 26. Mueller, P. & Hinnebusch, A. G. (1986) Cell 45, 201-207. protein, performs the 27. Werner, M., Feller, A., Messenguy, F. & Pierard, A. (1987) first step in the transcription of most eukaryotic genes. Cell 49, 805-813. We thank Audree Fowler and Janice Bleibaum for determining the 28. Gribskov, M., McLachlan, A. D. & Eisenberg, D. (1987) Proc. amino-terminal sequence ofthe TFIID protein, Bill Kimmerly for the Natl. Acad. Sci. USA 84, 4355-4358. gift of a yeast genomic library, Tom Sutherland for the timely and 29. Pearson, W. R. & Lipman, D. J. (1988) Proc. Natl. Acad. Sci. accurate synthesis of oligonucleotides, Roland Leuthy and David USA 85, 2444-2448. Eisenberg for protein sequence analysis, Haixin Xu for providing 30. Parraga, G., Horvath, S. J., Eisen, A., Taylor, W. E., Hood, yeast RNA preparations, and Qiang Zhou for innumerable contri- L., Young, E. L. & Klevit, R. E. (1988) Science 241, 1489- butions. This work was supported by a postdoctoral fellowship 1495. (PF2715) from the American Society to M.C.S., by a post- 31. Pabo, C. & Sauer, R. (1984) Annu. Rev. Biochem. 58, 293-321. doctoral fellowship (J-3-89) from the California Division of the 32. Landschulz, W. H., Johnson, P. F. & McKnight, S. L. (1988) American Cancer Society to C.C.K., and by National Cancer Science 240, 1759-1764. Institute Grants CA41062 and CA25235. 33. Kretsinger, R. H. (1987) Cold Spring Harbor Symp. Quant. 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