Mechanism of action of an acidic transcriptional activator in vitro

Lin, Yaojian; Green, Michael R.

doi:10.1016/0092-8674(91)90321-o

Cited by 537 publications

(431 citation statements)

References 44 publications

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“…ld, lanes 3, 5, 7, and 9). Previous studies have shown that the VP16 activation domain can bind TFIIB (65) and two subunits of TFIID, TBP (103) and TAF40 (30). Consistent with these studies, we found that GST-VP16 columns abound TFIID and TFIIB activities in addition to TFIIH activity (data not shown).…”

Section: Interaction Of the Vp16 Activation Domain With Tfiihsupporting

confidence: 80%

“…These include activation domains in VP16 (30,66), Epstein-Barr virus R (70), and human c-Rel (118) and CTF (57), as well as COUP-TF and other members of the nuclear receptor superfamily (1,49 (112,114). Other experiments indicate that a direct interaction of the activator with TBP and/or TFIIB leads to recruitment of TFIIB into the preinitiation complex (11,57,65,66,105). When the DNA template contains multiple activator-binding sites, acidic activators also stimulate a subsequent step in the assembly of the preinitiation complex involving recruitment of the other general initiation factors and RNA polymerase II (11).…”

mentioning

confidence: 99%

“…To further purify this TFIIH, 15 ml of bound proteins in 0.1 M KCl buffer C was loaded onto a 1-ml phosphocellulose column (89 Affinity chromatography. The GST, GST-VP16, GST-VP16FP442, GST-VP16A456, GST-VP16A456FP442 and GST-p53 (4-mg/ml each) affinity columns were prepared by immobilizing GST or GST fusion proteins on glutathione-Sepharose beads as previously described (65 the reaction mixtures. Transcripts were initiated at the adenovirus type 2 major late promoter on the plasmid pML(C2AT)-50 (93) in reaction mixtures containing bacterially expressed recombinant TFIIB (100 ng/ml), TBP (38 ng/ml), TFIIEa (100 ng/ml), TFIIE, (100 ng/ml), RAP30 (50 ng/ml) and RAP74 (50 ng/ml), a TFIIA/J fraction (1 RI), a TFIIH fraction (2 ,ul), and calf thymus RNA polymerase II (2.5 ,ug/ml).…”

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confidence: 99%

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Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

Xiao¹,

Pearson²,

Coulombe³

et al. 1994

Mol. Cell. Biol.

339

274

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Acidic transcriptional activation domains function well in both yeast and mammalian cells, and some have been shown to bind the general transcription factors TFIID and TFIIB. We now show that two acidic transactivators, herpes simplex virus VP16 and human p53, directly interact with the multisubunit human general transcription factor TFIHH and its Saccharomyces cerevisuze counterpart, factor b. The VP16-and p53-binding domains in these factors lie in the p62 subunit of TFIIH and in the homologous subunit, TFB1, of factor b. Point mutations in VP16 that reduce its transactivation activity in both yeast and mammalian cells weaken its binding to both yeast and human TFIIH. This suggests that binding of activation domains to TFIIH is an important aspect of transcriptional activation.Accurate transcription in vitro by human RNA polymerase II involves the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF (RAP30 and RAP74), TFIIH (also known as BTF2), and TFIIJ (reviewed in references 13 and 121). Beginning with TFIID, which recognizes the TATA boxes present in many promoters for RNA polymerase II, these factors and RNA polymerase II can be assembled in a defined order onto a promoter (5). TFIIH and TFIIJ are the last of these factors to bind to an assembling initiation complex (12,14,26), and TFIIH, also known as BTF2, is the only factor known to possess associated enzymatic activities. These include an ATP-dependent DNA helicase activity (95, 96) and a protein kinase activity that can phosphorylate the carboxyterminal heptapeptide repeat domain (CTD) of the largest subunit of RNA polymerase 11 (12, 21,68 herpes simplex virus transactivator VP16 (107) and in the N-terminal 73 amino acids of the mammalian tumor suppressor protein p53 (23). The p53 protein has a site-specific DNA-binding domain in its carboxy-terminal portion (101). VP16 does not, by itself, bind specifically to DNA, but instead its amino-terminal portion binds DNA in association with mammalian factors, including the POU homeodomain protein Oct-1 that recognizes octamer sequences in DNA (28,60,102). When fused to the DNA-binding domain of GAL4, the VP16 and p53 activation domains stimulate transcription in yeast and human cells of a gene bearing GAL4-binding sites (9,16,23,74,87,92). These observations imply that common mechanisms for transcriptional activation by acidic activators may exist in fungi and mammals.Many activation domains have been shown to interact with the TATA-box-binding protein (TBP) subunit of TFIID. These include the highly acidic activation domains in VP16 (50,103), p53 (10,67,72,84,97,108),118), and E2F-1 (37), as well as other kinds of activation domains found in the adenovirus activator ElA (48, 62), the Epstein-Barr virus proteins Zta (64) and R (70), the human T-cell leukemia virus type 1 (HTLV-1) activator Taxl (8), the transactivator Tat of human immunodeficiency virus type 1 (HIV-1) (53), and human c-Fos and c-Jun (85), PU-1 (38), and Spl (20). In certain cases, reduced binding of TBP by activation domains wit...

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Section: Interaction Of the Vp16 Activation Domain With Tfiihsupporting

confidence: 80%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

Xiao¹,

Pearson²,

Coulombe³

et al. 1994

Mol. Cell. Biol.

339

274

View full text Add to dashboard Cite

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“…Data suggesting that the VP16 activation domain can specifically interact with at least three distinct components of the transcription initiation complex in vitro, i.e., TBP, TFIIB, and the 40-kDa TAF (TAF40), have been presented (15,23,27,44). To address whether the results obtained with the VP16 activation domain are predictive for the minimal AAM defined in this work, we examined whether either TBP or TFIIB was also capable of binding to the AAM.…”

Section: Discussionmentioning

confidence: 99%

“…Recent studies have demonstrated that the activation domain of VP16 interacts with the basal transcription factors TBP and TFIIB in vitro, thus implicating one or both factors as important in vivo targets for acidic activators (23,27,44). The identification of a potent AAM derived from RelA afforded us the opportunity to examine the interaction of minimal core activation sequences with their putative target proteins.…”

mentioning

confidence: 99%

Mutational analysis of the transcription activation domain of RelA: identification of a highly synergistic minimal acidic activation module.

Blair¹,

Bogerd²,

Madore³

et al. 1994

Mol. Cell. Biol.

107

124

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The potent C-terminal activation domain of the RelA(p65) subunit of the cellular transcription factor NF-KB is shown to contain several discrete acidic activation modules. These short, -11-amino-acid modules were able to give rise to only a low level of transcription activation when fused to the GAL4 DNA-binding domain as monomers. However, dimers and higher-order multimers activated the transcription of minimal promoter elements as effectively as the full-length RelA or VP16 activation domain. Therefore, this 11-aminoacid ReIA-derived acidic module appears to contain all of the sequence information required to fully activate a target promoter element as long as it is presented in a form that permits functional synergy. Critical primary sequence requirements for acidic activation module function included a core phenylalanine residue and flanking bulky hydrophobic residues. Overall negative charge was necessary but not sufficient for function. While dimeric forms of the 11-amino-acid acidic activation module bound to either TFIIB or TATA-binding protein efficiently in vitro, a similarly charged peptide lacking the core phenylalanine residue failed to interact.Overall, these data demonstrate that the biological activity of the RelA activation domain is dependent on acidic activator sequences that are closely comparable to those detected in the activation domain of the viral VP16 regulatory protein. We hypothesize that the ability of these acidic activators to specifically interact with multiple components of the transcription initiation complex likely underlies the dramatic functional synergy exhibited by this class of activation domains in vivo.

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Localization of the 12-O-tetradecanoylphorbol-13-acetate response of the human ornithine decarboxylase promoter to the TATA box

1996

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In a previous study, we narrowed the region of the human ornithine decarboxylase (ODC) promoter responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA) to nt -42 to +54 around the transcription initiation site (Kim YJ, Pan H, Verma AK, Mol Carcinog 10:169-179, 1994). Here we report defining the role of the TATA box in TPA-induced transcription from the -42/+54 ODC promoter fragment. A transversion mutation at the third position of the TATA box (TATAAGT-->TAAAAGT) reduced TPA responsiveness of the reporter construct -42/+54 ODC-Luc by 49%. Electrophoretic mobility shift assays (EMSAs) using HeLa cell nuclear protein extracts revealed no differences in the binding pattern between the natural -42/+54 ODC promoter element and the -42/+54 ODC promoter element containing the T-->A mutation. However, antibodies to the general transcription factor TFIIB disrupted the DNA-protein complexes normally formed with the -42/+54 ODC promoter element in EMSAs. A consensus TATA box oligonucleotide formed two bands, with the faster mobility band displaying enhanced binding with nuclear protein extracts from TPA treated cells. Furthermore, incubation of HeLa cell nuclear extracts with an oligonucleotide containing the ODC TATA box also caused formation of two specific bands in EMSA. Both bands exhibited augmented binding to nuclear proteins from TPA-treated cells. Introduction of the T-->A transversion mutation in the ODC TATA oligonucleotide eliminated binding of the faster migrating band formed with the natural ODC TATA oligonucleotide. These results indicate that TPA modulation of the general transcription machinery may play a role in the TPA-activated transcription of the human ODC promoter.

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Mechanism of action of an acidic transcriptional activator in vitro

Cited by 537 publications

References 44 publications

Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

Mutational analysis of the transcription activation domain of RelA: identification of a highly synergistic minimal acidic activation module.

Localization of the 12-O-tetradecanoylphorbol-13-acetate response of the human ornithine decarboxylase promoter to the TATA box

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