The tumor suppressor protein p53 is a transcriptional regulator that enhances the expression of proteins that control cellular proliferation. The multisubunit transcription factor IID (TFIID) is thought to be a primary target for site-specific activators of transcription. Here, a direct interaction between the activation domain of p53 and two subunits of the TFIID complex, TAFII40 and TAFII60, is reported. A double point mutation in the activation domain of p53 impaired the ability of this domain to activate transcription and, simultaneously, its ability to interact with both TAFII40 and TAFII60. Furthermore, a partial TFIID complex containing Drosophila TATA binding protein (dTBP), human TAFII250, dTAFII60, and dTAFII40 supported activation by a Gal4-p53 fusion protein in vitro, whereas TBP or a subcomplex lacking TAFII40 and TAFII60 did not. Together, these results suggest that TAFII40 and TAFII60 are important targets for transmitting activation signals between p53 and the initiation complex.

The Origin Recognition Complex (ORC) is a six-protein assembly that specifies the sites of DNA replication initiation in S. cerevisiae. Origin recognition by ORC requires ATP. Here, we demonstrate that two subunits, Orc1p and Orc5p, bind ATP and that Orc1p also hydrolyzes ATP. ATP binding and hydrolysis by Orc1p are both regulated by origin DNA in a sequence-specific manner. ATP binding to Orc1p, but not ATP hydrolysis, is responsible for the ATP dependence of the ORC-origin interaction, indicating that ATP is a cofactor that locks ORC on origin DNA. These data demonstrate that occupancy of the Orc1p ATP-binding site has a profound effect on ORC function and that ATP hydrolysis by Orc1p has the potential to drive transitions between different functional states of ORC.

The Saccharomyces cerevisiae origin recognition complex (ORC) is bound to origins of DNA replication throughout the cell cycle and directs the assembly of higher-order protein±DNA complexes during G 1 . To examine the fate of ORC when origin DNA is unwound during replication initiation, we determined the effect of single-stranded DNA (ssDNA) on ORC. We show that ORC can bind ssDNA and that ORC bound to ssDNA is distinct from that bound to doublestranded origin DNA. ssDNA stimulated ORC ATPase activity, whereas double-stranded origin DNA inhibited the same activity. Electron microscopy studies revealed two alternative conformations of ORC: an extended conformation stabilized by origin DNA and a bent conformation stabilized by ssDNA. Therefore, ORC appears to exist in two distinct states with respect to its conformation and ATPase activity. Interestingly, the effect of ssDNA on these properties of ORC is correlated with ssDNA length. Since double-stranded origin DNA and ssDNA differentially stabilize these two forms of ORC, we propose that origin unwinding triggers a transition between these alternative states.

Regulated transcription of protein encoding genes in eukaryotes by RNA polymerase II requires a multitude of factors including transcriptional activators that bind DNA to enhance transcription in a gene-specific manner and basal factors (transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) that are required for promoter-specific initiation (for reviews, see refs. 1 and 2). Of these factors, TFIID specifically recognizes and binds to "TATA boxes" found within many promoters. We now know that TFIID is a multisubunit complex containing the TATA binding protein (TBP) and at least eight TBP-associated factors (TAFs; refs. 3-5; for review, see ref. 6). It was found that recombinant TBP can substitute for TFIID in directing basal levels of transcription in vitro (7, 8). However, transcription driven by TBP alone is not responsive to transcriptional activators. Thus TAFs appear to represent a unique class of factors in that they are not necessary to direct basal transcription from TATA-containing promoters but are required for transcriptional activation.This observation (8) led to the proposal that some of the TAFs in the TFIID complex would directly contact activators and basal factors thereby mediating signals from activators to the basal transcription machinery and, furthermore, that different classes of activation domains (such as acidic, glutaminerich, proline-rich, or isoleucine-rich) would contact distinct TAFs within the TFIID complex. This laboratory has now obtained (9-13) evidence for a number of interactions between TAFs and transcriptional activators and between TAFs and other basal transcription factors. Direct evidence for the function of TAFs as coactivators was obtained when recom-The 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. §1734 solely to indicate this fact.binant TAFs and TBP were assembled into partial TFIID complexes that could support transcriptional activation in vitro by a number of activators (11,12). For example, a partial complex consisting of the 250-kDa subunit of human TFIID (hTAFI1250), Drosophila (d) TBP, dTAFI150, and dTAFlillO can replace a crude TFIID fraction in mediating transcriptional activation by Spl (11). However, all of our studies examining the activity of partial TFIID complexes have used Drosophila coactivators. It was therefore important to test the generality of the specific TAF-activator interactions in mediating transcriptional activation by using the human subunits.We have previously reported the cloning and characterization of dTAF1140 (10). This 40-kDa subunit of dTFIID was found to specifically interact with the C-terminal 39-amino acid activation domain of viral protein 16 (VP16) (termed VP16c) and with the basal transcription factor TFIIB. Here we extend our analysis of coactivators for VP16 by cloning and characterizing the human homologue of dTAFI40, hTAFI132.J We have expressed recombinant hTAF1132 in several forms and ...