in a solution of 10 mM 3-[cyclohexylamine]-1- propane-sulfonic acid (CAPS)-NaOH (pH 11.0) and 10% methanol (the TFB1 protein does not transfer efficiently in buffers commonly used for transfer to nitrocellulose). The membrane was incubated with a 1:200 dilution of preimmune or immune serum, and bound antibodies were visu- alized by means of alkaline phosphatase-conju- gated secondary antibodies. 28. Preimmune or immune serum was mixed with an equal volume of Affi-Gel-protein A beads (Bio- Rad), the beads were washed, and the antibodies were covalently cross-linked to the protein A with dimethyl pimelimidate (24). The columns were washed in elution buffer [0.1 M glycine-HCI (pH 3), glycerol (20%)] and equilibrated in binding buffer [20 mM Hepes (pH 7.5), 0.1 M potassium acetate, glycerol (20%), 1 mM DTT, 1 mM EDTA, Triton X-100 (0.1%), and protease inhibitors (6)]. The factor b used in Fig. 3B was a partially purified preparation obtained by fractionation of a yeast whole-cell extract on Biorex 70 (Bio-Rad) and DEAE-Sephacel (Pharmacia) columns (25). The preparation (1.5 ml) was applied to a preim- mune or immune antibody column (0.5 ml), and the flow through was reapplied to the column five times. The column was washed with 10 volumes of binding buffer followed by 50 volumes of bind- ing buffer containing 0.8 M potassium acetate and 50 volumes of binding buffer containing Triton X-100 (0.5%) and sodium deoxycholate (0.5%). Bound proteins were eluted with several 0.25-mI portions of elution buffer. Each eluted fraction was immediately neutralized by the addition of 15 g1 of 2 M tris-acetate, pH 8.0. The eluted fractions were concentrated by trichloroacetic acid precipitation, analyzed by SDS-PAGE, and stained with Coomassie blue. The same starting material and immunoaffinity procedure were used in Fig. 4A, except that the detergent wash was omitted. In Fig. 4B, the fraction loaded on the column was the same as in Fig. 3A, and the immunoaffinity proce- dure was the same as in Fig. 4A. 29. A. M. Edwards et al., Proc. Nati. Acad. Sci. U.S.A. 87, 2122 (1990). 30. We thank J.-M. Egly and co-workers and J. Con- away and R. Conaway and co-workers for sharing unpublished results; N. Shavit for help with affinity labeling; A. Edwards for supplying RNA polymer- ase 11; H. Goodson for help with tetrad analysis; and P. Patek and S. Martinez for synthesizing oligonucleotides. O.G. was supported by a Weiz- mann postdoctoral fellowship and by an Ameri- can Cancer Society (California Division) postdoc- toral fellowship. W.J.F. was supported by a Med- ical Research Council of Canada studentship. This research was supported by NIH grant GM- 36659 to R.D.K. 12 March 1992; accepted 22 June 1992 Cloning of the 62-Kilodalton Component of Basic Transcription Factor BTF2 Laurent Fischer, Matthieu Gerard, Christian Chalut, Yves Lutz, Sandrine Humbert, Masamoto Kanno, Pierre Chambon, Jean-Marc Egly* Cloning of the mammalian basic transcription factors serves as a major step in under- standing the mechanism of transcription initiation. The 62-kilodafton component (p62) of one of these transcription factors, BTF2 was cloned and overexpressed. A monoclonal antibody to this polypeptide inhibited transcription in vitro. Immunoaffinity experiments demonstrated that the 62-kilodafton component is closely associated with the other poly- peptides present in the BTF2 factor. Sequence similarity suggests that BTF2 may be the human counterpart of RNA polymerase 11 initiation factor b from yeast. In vitro transcription of genes requires RNA polymerase B (II) and transcription factors TFIIA, TFIIB, TFIID (1), TFIIE, TFIIF (2-4), and BTF2 (5). The BTF2 transcription factor was purified and found to coelute or to cosediment with five poly- peptides ranging from 35 to 90 kD after five chromatographic steps and one glycerol gra- dient (5). Transcription factor BTF2 is absolutely required for accurate in vitro transcription from a minimal promoter (containing the cap site and the TATA box) and sediments with a molecular size of 250 kD (5). Other transcription factors have also been identified and purified and Laboratoire de Genetique Moleculaire des Eucaryotes du Centre National de la Recherche Scientifique, Unit6 184 de Biologie Mol6culaire et de Genie Gene- tique de lInstitut National de la Sant6 et de la Recher- che Medicale, Facult6 de M6decine, 11 rue Humann, 67085 Strasbourg C6dex, France. *To whom correspondence should be addressed. include TFIIG, TFIII (6), TFIIH (7), and 8 (8). It is unclear whether BTF2 corresponds to one or more of these factors (5-8). We now report the cloning and properties of the 62-kD component (p62) of BTF2. We designed three degenerate oligonu- cleotide probes on the basis of the se- quences of tryptic peptides from the purified p62 and used them for the screening of a HeLa cell cDNA library (9). Three inde- pendent cDNA clones were isolated and sequenced. All of them contained a pre- dicted open reading frame (ORF) encoding a polypeptide of 548 amino acids with a calculated molecular size of 62.030 kD and an isoelectric point (pI) of 8.82 (Fig. 1). The encoded polypeptide contained the three oligopeptides that we microse- quenced. We found no similarity with any of the sequences present in protein and nucleic acid databases. No known DNA binding motifs, kinase motifs, or nucleotide binding sites were found. When overex- pressed in Escherichia cob (10) or in insect cells (11), the recombinant polypeptide (rp62) had the same electrophoretic mobil- ity on SDS-polyacrylamide gel electropho- resis (PAGE) as the p62 from BTF2. The recombinant protein from either source did not substitute for the BTF2 activity in a HeLa cell in vitro transcription system lack- ing BTF2 (12), and no stimulation was observed when rp62 was added to an in vitro transcription system containing limit- ing amounts of BTF2. This result indicates that rp62 alone is not sufficient to restore BTF2 activity and that other BTF2 poly- peptides may be required. Several lines of evidence indicate that p62 is associated with BTF2 activity. First, a monoclonal antibody (M.Ab3c9) to pu- rified rp62 (13), recognized a 62-kD poly- peptide (p62) that cofractionated with the BTF2 activity throughout the purification (Fig. 2, A to D) (5, 12, 14). Second, inhibition of in vitro transcription indicat- ed that p62 is a component of the BTF2 activity. Increasing amounts of either puri- fied M.Ab3c9 or a control antibody M.AbC (13) were incubated for 1 hour at Fig. 1. Analysis of the hu- 1 M A T S S E E V L L I V XK V R Q K K Q D G A L Y L M A E R I A W A P E G X D R man BTF2 p62 polypeptide 41 F T I S H M Y A D I X C Q K I S P E G X A K I Q L Q L V LH A G D T ? NK P HF S sequence. Positions of the 81 N E S T A V K E R D A V X R D LL Q L L P F K R K A NK E E L E E K NR M L Q E amino acids are indicated 121 D P V L F Q L Y K D L VV S Q v I S A E E F W A N R L N V N A T D S S S T S N H on the left. The three pep- 161 X Q D V G I S A AI F L A D V R P Q T D G C N G L R Y N L TS D I I E S I F R T Y tides that were microse- 201 P A V X K Y A E N V P H N M T E K E F M T R F F Q SHY F H R D R LN T G S K quenced from the purified 241 D L F A E C A X I D E X G L K T n V S L G V X N P LL D LT A LIE D K P L DE G 62-kD polypeptide are 281 Y G I S S V P S A S N S K S I K E N S N A A I I X R P M H H S A M V L AA G LR boxed. Two imperfect ami- no acid repats [KDLLO/ 321 X Q E A Q N E Q TS E P S N Mt D G M S G D A D C P Q P A V K R A K |L Q E S I E Y no acid repeats [KDLLQ/ 31ELKNVTANKSRYGTISQASD QLLPK (residues 93 to 361 E D L GK KN N S V K T I A L W L X K S D R Y Y H G PT P I Q S L Q Y A T S Q D I QLLPK (residues 93 to 102) and LSSSAIASSTI 401 I N S F Q S I R Q E M E A Y T P K L T Q V L S S S A A SS T I T A L S P G G A L (residues 422 to 431)] are 441 Q G G T Q Q A I N Q M V P N D I Q S E L K H L Y V A V G EL L R H F S CF P shown by inverted bold ar- 481 V N T P F L E E K V V K MX S N L E R r Q V T K LC P F Q E K I RR QYL S TN rows. The nucleotide se- 521 L V S H I E E M L Q T A Y N K L H T W Q S R R L M K X T quence of the BTF2 p62 subunit can be found in GenBank under the accession number M95809. Abbreviations for the amino acid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lie; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; 0, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. SCIENCE * VOL. 257 * 4 SEPTEMBER 1992 p. P,' ............................ .---- W01 M11A Y0 manno. 1392 on March 26, 2016 Downloaded from on March 26, 2016 Downloaded from on March 26, 2016 Downloaded from on March 26, 2016 Downloaded from