ABSTRACTp53 activates transcription of genes with a p53 response element, and it can repress genes lacking the element. Here we demonstrate that wild-type but not mutant p53 Inhibits transcription in a HeLa nuclear extract from minimal promoters. Wild-type but not mutant p53 binds to human TATA-binding protein (TBP). p53 does not bind to yeast TBP, and it cannot Inhibit tscription in a HeLa extract where yeast TBP subsitutes for hun TBP. These results suggest a model in which p53 binds to TBP and interferes with transcriptional initiation.The p53 gene product functions as a transcription factor. It can activate transcription when bound to a promoter through a heterologous DNA-binding domain (1,2). Wild-type p53 (p53wt) binds to DNA sequences termed p53 response elements (3, 4), and when these binding sites are adjacent to a minimal promoter, they stimulate expression in a p53-dependent fashion (5-8). p53wt also negatively regulates a variety of genes that lack a p53 response element, including the c-fos, c-jun, retinoblastoma, interleukin 6, and proliferating cell nuclear antigen genes as well as the p53 gene itself (9)(10)(11)(12). Expression of a class I major histocompatibility complex gene (9) and the Ha-rasl gene (13,14), however, are reportedly neither activated nor repressed by p53wt. Thus, it appears that p53wt may exert positive or negative effects on the expression of some but not all genes, and this may form the mechanistic basis for its ability to regulate cell proliferation.Here we show that p53wt, but not mutant p53 (p53mt), inhibits transcription in nuclear extracts from minimal promoters. Furthermore, p53wt can bind directly to the human TATA-binding protein (TBP). These results suggest that p53 functions as part of the transcriptional machinery, regulating transcription. MATERIALS AND METHODSPlasmids, Chioramphenicol Acetyltranserase (CAT) Assays, and Protein Purifiation. p11-4 encodes a murine pS3wt (m-p53wt) cDNA and pSVKH215 contains a m-p53mt cDNA with a four-amino acid insert at residue 215; both are controlled by the simian virus 40 early promoter (15). pc53-CIN encodes a human p53wt (h-pS3wt) gene containing introns 2-4, under control of the cytomegalovirus immediate-early promoter, and pc53-Cx22AN is identical to pc53-CIN except that its coding region contains an Arg-175 to His substitution (16). pTICAT (17), p50-2 (8), and pMLTATA (18) have been described. h-p53wt and His-175 h-p53mt cDNAs were subcloned into pETild DNA (19) to create pET-p53wt and pET-p53mt. Plasmids 12028The 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.

It is generally believed that shutting down the kinase activity of BCR-ABL by imatinib will completely inhibit its functions, leading to inactivation of its downstream signaling pathways and cure of the disease. Imatinib is highly effective at treating human Philadelphia chromosome-positive (Ph ؉ ) chronic myeloid leukemia (CML) in chronic phase but not Ph ؉ B cell acute lymphoblastic leukemia (B-ALL) and CML blast crisis. We find that SRC kinases activated by BCR-ABL remain fully active in imatinib-treated mouse leukemic cells, suggesting that imatinib does not inactivate all BCR-ABLactivated signaling pathways. This SRC pathway is essential for leukemic cells to survive imatinib treatment and for CML transition to lymphoid blast crisis. Inhibition of both SRC and BCR-ABL kinase activities by dasatinib affords complete B-ALL remission. However, curing B-ALL and CML mice requires killing leukemic stem cells insensitive to both imatinib and dasatinib. Besides BCR-ABL and SRC kinases, stem cell pathways must be targeted for curative therapy of Ph ؉ leukemia.dasatinib ͉ imatinib ͉ SRC kinases

The structures of the ligand-binding domains (LBD) of the wildtype androgen receptor (AR) and the T877A mutant corresponding to that in LNCaP cells, both bound to dihydrotestosterone, have been refined at 2.0 Å resolution. In contrast to the homodimer seen in the retinoid-X receptor and estrogen receptor LBD structures, the AR LBD is monomeric, possibly because of the extended C terminus of AR, which lies in a groove at the dimerization interface. Binding of the natural ligand dihydrotestosterone by the mutant LBD involves interactions with the same residues as in the wildtype receptor, with the exception of the side chain of threonine 877, which is an alanine residue in the mutant. This structural difference in the binding pocket can explain the ability of the mutant AR found in LNCaP cells (T877A) to accommodate progesterone and other ligands that the wild-type receptor cannot.

The two forms of RNA polymerase H that exist in vivo, phosphorylated (HO) and nonphosphorylated (HA), were purified to apparent homogeneity from HeLa cells. The nonphosphorylated form preferentially binds to the preinitiation complex. RNA polymerase H in the complex was converted by a cellular protein kinase to the phosphorylated form.Purified RNA polymerase II cannot accurately initiate transcription from class II promoters in vitro unless it is supplemented with general transcription initiation factors (1-3). Seven human general transcription factors (TFIIA, -IIB, -IID, -lIE, -IIF, -IIG, and -IIH) that, together with RNA polymerase II are sufficient for specific transcription, have been identified (O.F. and D.R., unpublished results).Despite progress in the purification of the general transcription factors, determination of their individual activities and contributions to the formation of a transcriptioncompetent complex remains obscure. Complicating these analyses is the existence in eukaryotic cells of two different forms of RNA polymerase II, IIA and IIO, which differ in the level of phosphorylation of a highly conserved heptapeptide repeat present at the carboxyl terminus of the largest subunit (carboxyl-terminal domain; CTD) (4-6). The heptapeptide repeat is essential for viability (7-10); nevertheless, a third species ofRNA polymerase II lacking the CTD (IIB) has been observed in vitro (11)(12)(13)(14). Photoaffinity labeling experiments demonstrated that nascent RNA transcripts crosslink almost exclusively to the phosphorylated IIO form in vivo and in vitro (5,15,16), suggesting that it is the IIO polymerase that elongates RNA chains. Monoclonal antibodies against the nonphosphorylated IIA form inhibited specific transcription initiation (17, 18), suggesting that RNA polymerase IIA was more active than IIO during specific transcription initiation in vitro. Therefore, the CTD phosphorylation state may regulate the transition from initiation to elongation (19).We have purified human RNA polymerases IIO and IIA to apparent homogeneity and analyzed their roles in transcription. We demonstrate that the IIA form associates preferentially with the preinitiation complex where it is then converted by a cellular protein kinase to the phosphorylated 11O form. MATERIALS AND METHODSTranscription Factors, Transcription Reaction Mixtures, and DNA Binding Assays. Transcription reactions and DNA binding assays were performed as described (20). Transcription factor IIA (TFIIA) (P. Cortes and D.R., unpublished data), TFIIB (22), TFIIE (23), and TFIIF (24) were purified from HeLa cell nuclear extracts as described. TFIID was purified to homogeneity as described (20) Purification of the Phosphorylated (HO) and Nonphosphorylated (HA) Forms of RNA Polymerase H. RNA polymerase II was purified from HeLa cell nuclear pellets (7.5 x 1010 cells). Enzyme solubilization and chromatography on DE-52 were as described (26). Active fractions ofthe DE-52 column, between 0.2 and 0.3 M salt, were pooled (109 mg of protein, 170 ml...