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Human transcription factor TFIID, the TATA-binding protein, was partially purified to a form capable of associating stably with the TATA motif of the adenovirus major late promoter. Binding of the human and yeast TFIID to the TATA motif was stimulated by TFIIA. TFIIA is an integral part of a complex capable of binding other transcription factors. A complex formed with human TFIID and TFIIA (DA complex) was specifically recognized by TFIIB. We found that TFIIB activity was contained in a single polypeptide of 32 kDa and that this polypeptide participated in transcription and was capable of binding to the DA complex to form the DAB complex. Formation of the DAB complex required TFIIA, TFIID, and sequences downstream of the transcriptional start site; however, the DA complex could be formed on an oligonucleotide containing only the adenovirus major late promoter TATA motif. Using anti-TFIIB antibodies and reagents that affect the stability of a transcription-competent complex, we found that yeast and human TFIID yielded DAB complexes with different stabilities.The promoters of genes transcribed by RNA polymerase II are composed of different cis-acting sequence elements that participate in the regulation of expression (23). Two of these elements, the TATA motif, which is located approximately 30 nucleotides upstream of the transcriptional start site (1), and the initiator (34), which .encompasses the start site of transcription, are present in many genes. These two transcriptional control elements appear to be recognized by a complex set of transcription factors (for reviews, see references 24 and 30). The factors operating through these elements are required for transcription of all genes thus far studied, including those that do not contain a TATA motif, and therefore they have been classified as general or basal transcription factors. Of the five general transcription factors (TFIIA, -IIB, -IID, -IIE, and -IIF) that have been described, TFIID appears to be the only factor with an associated DNA-binding activity with specificity for the TATA motif (6,9,18,20,25,28,35). The other factors and RNA polymerase II appear to associate with promoter sequences primarily by protein-protein interactions. Indeed, it has been demonstrated that TFIIE and TFIIF can independently form a stable complex (the TFIIE/F complex) with RNA polymerase II (10, 11). Moreover, it has been suggested that a protein fraction containing TFIIE/F can form a complex with TFIIB (29).The human TFIID (hTFIID) has been difficult to purify. A breakthrough in the field was the discovery that yeast cells contained a TFIID activity (yTFIID) that could functionally substitute for the hTFIID in transcription systems reconstituted with all human factors (3,5). The yTFIID activity has been purified to homogeneity; these studies demonstrated that the yTFIID activity is contained in a single polypeptide of 27 kDa (20). The reported molecular weight of the yeast factor was confirmed with the isolation of the yTFIID gene (4,8,14,19,33). Isolation of the yTFIID gen...

The previously described transcription factor IIA (TFIIA) protein fraction was separated into two factors that affect transcription, TFIIA and TFIIJ. TFIIA was found to have a stimulatory effect, and TFIIJ was found to be required for transcription. The tein fraction is composed of at least two separate components, TFIIA and TFIIJ. We found that TFIIA acted to increase transcription, whereas transcription was dependent on TFIIJ. The TFIIA-mediated stimulation of transcription was dependent on the source of TFIID. TFIIA was found to have a stimulatory effect only when the transcription assays contained endogenous HeLa cell TFIID. Transcription assays reconstituted with recombinant yeast TBP (yTBP) or human TBP (hTBP) were independent of TFIIA. MATERIALS AND METHODSSpecific transcription assay and transcription factors. Transcription reactions and product analysis were performed as previously described (36). The DNA template used was ApML(C2AT), which contains the adenovirus major late promoter (Ad-MLP) sequences extending from -50 to +11 For assays measuring TFIIA activity, the TFIID protein fraction was used and the TFIIA protein fraction was omitted and replaced by column fractions. For assays measuring TFIIJ activity, h-or yTBP was used in lieu of TFIID and the TFIIE/F/H protein fraction was replaced by more purified factors (14a). The TFIIJ protein fraction used was derived from either the phosphocellulose 0.1 M KCI or TFIIG (45) protein fractions, as indicated in the figure legends. TFIIJ derived from the 0.1 M KCI fraction was purified as described below. TFIIJ derived from the TFIIG

Genes for immunoglobulins and T-ceil receptor are generated by a process known as V(D)J recombination. This process is highly regulated and mediated by the recombinatlon activating proteins RAG-1 and RAG-2. By the use of the two-hybrid protein interaction system, we isolated a human protein that specifically interacts with RAG-i. This protein is the human homologue of the yeast SRP1 ( Here we describe the human (h) and mouse (m) homologues of SRP1, we show their interaction with RAG-1 in yeast as well as in mammalian cells, and we map the regions responsible for the interaction in both proteins. The possible role of SRP1 in V(D)J recombination is discussed.MATERIALS AND METHODS Cels and Antibodies. 293T cells (19) were grown in Dulbecco's modified Eagle's medium containing 10o fetal bovine serum, penicillin, and streptomycin. Anti-influenza hemagglutinin (HA) monoclonal antibody (12CA-5) was from Berkeley antibody company. Anti-glutathione S-transferase (GST) antibodies were generated in rabbits and affinity purified. pcDNAI/amp (Invitrogen), p65/GST, and p50-HA expression vectors will be described elsewhere.DNA Constructs. A pEG202RAG-1 vector encoding fulllength RAG-1 was assembled from a BamHI/EcoRV PCR product, an EcoRV/Not I fragment from RAG-1/CDM8, and a Not I/BamHI pEG202 vector (15). Ligation of these three fiagments generated pEG202/RAG-1. The joining segments were sequenced and the expression of the fusion product RAG-1-LexA was tested by transformation in yeast followed by Western blotting. pEG202/RAG-1 was used to screen a human cDNA library. All the subcloning of RAG-1 and hSRP1 homologue was done by production of in-frame PCR firgments or by directly subcloning from the cDNA with short oligonucleotides to leave the protein in-frame. The junctions of all constructs were sequenced.For immunoprecipitation experiments, pcDNAI/amp (Invitrogen) was modified by introducing an oligonucleotide containing an initiation codon in-frame with the HA epitope Abbreviations: GST, glutathione S-transferase; V(D)J, variablediversity-joining region; h-, human; in-, mouse; HA, hemagglutinin; NLS, nuclear localization signal. 7633The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 11734 solely to indicate this fact.

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