A novel mediator between activator proteins and the RNA polymerase II transcription apparatus

“Protein was determined by the method of Bradford (1976) , using bovine serum albumin as a standard, and assays of P-galactosidase activity were performed as previously described (Miller, 1972; Guarente, 1983).”

“Assays of BGaiactosidase Activity in Vivo Plasmid DNAs were introduced into Saccharomyces cerevisiae strain 5C (a his3-A200 ura3-52) by a lithium acetate transformation procedure (ito et al., 1983).”

“…GAL4-VP16, comprising the DNA binding domain (amino acids 1-147) of GAL4 fused to the activation domain (carboxy-terminal 78 amino acids) of VP16 (Sadowski et al., 1988), and a yeast factor whose binding site is a thymidine-rich sequence from upstream of the DED7 gene (T-rich binding factor; Lue…”

“Protein was determined by the method of Bradford (1976), using bovine serum albumin as a standard, and assays of P-galactosidase activity were performed as previously described (Miller, 1972; Guarente, 1983).”

“One line of evidence comes from alterations in the DNAase I footprint of partially purified HeLa TFIID on the adenovirus major late and E4 promoters caused by activator protein binding (Sawadogo and Roeder, 1985b; Horikoshi et al., 1988a, 1988b).”

“For example, herpes viral protein 16 (VP16), an activator of immediate early gene expression in herpes virus-infected cells, causes a severalfold reduction in the level of transcription of an unrelated gene (Triezenberg et al., 1988).”

“A single copy plasmid for expression of GAL4VP16 from the ADH7 plromoter, pCEN4:G4VPlG, was created by the insertion of the Kpnl fragment containing CEN4 sequences from pCZA into the unique Xbal site of pMA424 (gift of J. Ma and M. Ptashne; Ma and Ptashne, 1987) by blunt end ligation, leaving the 2pm origin intact but…”

“Similarly, high levels of the activator GAL4 in yeast cells diminish expression of genes lacking GAL4 binding sites as much as 5fold (Gill and Ptashne, 1988).”

“In the case of the vertebrate estrogen, progesterone, and glucocorticoid receptors, which activate responsive genes in the presence of hormone, various pairwise combinations of both transfected and endogenous receptor genes exhibit interference in a hormone-dependent fashion (Meyer et al., 1989).”

“A similar inhibition of activation by the GRFI binding EN07 UAS was observed.”

“Possibly, the factor that binds to this element has a higher affinity than GAL4-VP16 for the component mediating activation, or alternatively, this element may activate transcription by a different mechanism (Struhl, 1985).”

“One line of evidence comes from alterations in the DNAase I footprint of partially purified HeLa TFIID on the adenovirus major late and E4 promoters caused by activator protein binding (Sawadogo and Roeder, 1985b; Horikoshi et al., 1988a, 1988b).”

“This observation of inhibition by GAL4-VP16 in vitro has been confirmed by others (Berger et al., 1990).”

“A yeast TATA binding protein purified according to published methods (Buratowski et al., 1988) and purified yeast RNA polymerase II (Edwards et al.”

“A similar inhibition of activation by the GRFI binding EN07 UAS was observed.”

“One line of evidence comes from alterations in the DNAase I footprint of partially purified HeLa TFIID on the adenovirus major late and E4 promoters caused by activator protein binding (Sawadogo and Roeder, 1985b; Horikoshi et al., 1988a, 1988b).”

“(The slight activity of the GAL70 UAS in the absence of GAL4-VP16 may be due to incomplete catabolite repression of endogenous GAL4 function in this yeast strain, or to the recognition of the UAS sequence by other factors [Fedor et al., 1988; Chasman et al., 19901.)”

“Similar experiments were performed with HeLa TFIIA, TFIIB, TFIID, and TFllElF (Reinberg et al., 1987) and with rat liver transcription factors a, Pr, 6, E, and T (Conaway et al.”

“Possibly, the factor that binds to this element has a higher affinity than GAL4-VP16 for the component mediating activation, or alternatively, this element may activate transcription by a different mechanism (Struhl, 1985).”

“(C) Transcription of p(DED48)aCG- as in (A) in the presence of 100 ng of the GAL4 binding oligonucleotide 3GALlO (solid diamonds) (Chasman et al., 1989) or a control oligonucleotide, SPT2 (dotted open squares) (Buchman and Kornberg.”

“, 1988), and a yeast factor whose binding site is a thymidine-rich sequence from upstream of the DED7 gene (T-rich binding factor; Lue et al., 1989a; Buchman and Kornberg, 1990).”

“Preparation and Fractionation of Nuclear Extracts Yeast nuclear extracts were prepared from strain BJ926 essentially as described (Lue and Kornberg, 1987; Lue et al., 1989a), with the following modifications.”

“5 mglml) was purified by procedures that maintained the integrity of the largest subunit (Edwards et al., 1990).”

“Ptashne; Ma and Ptashne, 1987) by blunt end ligation, leaving the 2pm origin intact but imposing centromteric regulation of copy number (Tschumper and Carbon, 1983).”

“…there was little effect of the GAL70 UAS, and the fNO7, DED7, and d(T)s9 UAS elements stimulated expression to the same extent as reported previously (compare levels of 6-galactosidase activity for these UAS elements and that for no UAS in Table 1; Buchman et al., 1988b; Lue et al., 1989a).”

“…there was little effect of the GAL70 UAS, and the fNO7, DED7, and d(T)s9 UAS elements stimulated expression to the same extent as reported previously (compare levels of 6-galactosidase activity for these UAS elements and that for no UAS in Table 1; Buchman et al., 1988b; Lue et al., 1989a).”

“The amount of each purified protein tested ranged from about 0.05 to 4 times that required for maximal basal transcription in a reconstituted yeast system (Flanagan et al., 1990).”

“, 1987) and with rat liver transcription factors a, Pr, 6, E, and T (Conaway et al., 1990).”

“Similar experiments were performed with HeLa TFIIA, TFIIB, TFIID, and TFllElF (Reinberg et al., 1987) and with rat liver transcription factors a, Pr, 6, E, and T (Conaway et al., 1990).”

“A second activity, termed mediator of transcriptional activation and originally demonstrated in yeast, confers a response to activator proteins upon a system reconstituted with TBP and is distinct from all the basal factors (Kelleher et al., 1990; Flanagan et al., 1991).”

“…purified recombinant TBP suggest instead that activator proteins and TBP may not interact directly but require intermediary proteins, or coactivators, to promote transcriptional activation (Berger et al., 1990; Hoey et al., 1990; Kelleher et al., 1990; Peterson et al., 1990; Pugh and Tjian, 1990).”

“However, the widespread belief in a direct mechanism was finally challenged by analysis of squelching in a crude yeast pol II transcription system in vitro [ 6 ].”

“Evidence for a transcriptional mediator came from squelching experiments, defined by the ability of one activator to inhibit transcription by another activator (138, 244).”