Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro.

Chasman, Daniel I.; Leatherwood, Janet; Carey, Michael; Ptashne, Mark; Kornberg, Roger D.

doi:10.1128/mcb.9.11.4746

Cited by 177 publications

(108 citation statements)

References 29 publications

(31 reference statements)

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“…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.…”

mentioning

confidence: 78%

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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mentioning

confidence: 78%

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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“…Transformations were carried out by a variation of the lithium acetate procedure previously described (32), and transformants were grown on selective VOL. 13,1993 on May 12, 2018 by guest http://mcb.asm.org/ Downloaded from minimal media containing 2% glucose. For studies involving YJO-Z it was necessary to transfer transformants to selective minimal media containing 2% glycerol and 3% lactic acid to induce lacZ expression.…”

Section: Methodsmentioning

confidence: 99%

Conservation of transcriptional activation functions of the NF-kappa B p50 and p65 subunits in mammalian cells and Saccharomyces cerevisiae.

Moore¹,

Ruben²,

Rosen³

1993

Mol. Cell. Biol.

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The NF-KB transcription factor complex is composed of a 50-kDa (p50) and a 65-kDa (p65) subunit. Both subunits bind to similar DNA motifs and elicit transcriptional activation as either homo-or heterodimers. By using chimeric proteins that contain the DNA binding domain of the yeast transcriptional activator GALA and subdomains of p65, three distinct transcriptional activation domains were identified. One domain was localized to a region of 42 amino acids containing a potential leucine zipper structure, consistent with earlier reports. Two other domains, both acidic and rich in prolines, were also identified. Of perhaps more significance, the same minimal activation domains that were functional in mammalian cells were also functional in the yeast Saccharomyces cerevisiae. Coexpression of the NF-KcB inhibitory molecule, IKB, reduced the transcriptional activity of p65 significantly, suggesting the ability of IKB to function in a similar manner in S. cerevisiae. Surprisingly, while the conserved rel homology domain of p65 demonstrated no transcriptional activity in either mammalian cells or S. cerevisiae, the corresponding domain in p50 was a strong transcriptional activator in S. cerevisiae. The observation that similar domains elicit transcriptional activation in mammalian cells and S. cerevisiae demonstrates strong conservation of the transcriptional machinery required for NF-cB function and provides a powerful genetic system to study the transcriptional mechanisms of these proteins.Nuclear factor KB (NF-KB), originally described as a factor involved in tissue-specific control of the mouse K light-chain enhancer (65,66), is now known to be involved in the inducible expression of a wide range of genes (2, 6, 44). The KB DNA binding site has been identified in the regulatory elements of cytokine, cytokine receptor, major histocompatibility complex class I and II antigens, inflammatory and acute-phase response genes, and several viral enhancer elements (2, 6, 44). DNA binding activity appears to be constitutive in mature B cells (66) and in activated macrophages (24). However, in most other cells NF-KB is complexed with an inhibitory molecule (IKB) in the cytoplasm (3,4,21). Sequestration in the cytoplasm is thought to involve masking of the nuclear localization signal (8). Upon activation by a variety of stimuli, including mitogens, cytokines, and various viral proteins (2, 6, 44), NF-KB shuttles to the nucleus, a process thought to involve phosphorylation of IKB (21,68). Initial studies demonstrated that IKB inhibits binding of the p65 homodimer and the p5O/p65 heterodimer through interaction with p65 (5, 21, 54, 78). However, recent studies suggest that both subunits can interact with IKB (8).Biochemical studies have shown that the major form of NF-KB is composed of a heterodimer of a 50-kDa (p50) and a 65-kDa (p65) subunit (5). Both subunits of NF-KB have been cloned, and sequence analyses demonstrate strong similarity in the amino-terminal 300 amino acids to the rel proto-oncogene product (22,38,54,58), origi...

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“…Purified Sp1 was obtained from Promega (Madison, WI). Gal4-VP16 was purified as described previously (19). Transcription reactions with NF-B p65 and Sp1 were carried out with a DNA template containing the IRF-1 promoter region (from Ϫ1312 to ϩ39 relative to the RNA start site) (20).…”

Section: Methodsmentioning

confidence: 99%

Transcriptional Analysis of Chromatin Assembled with Purified ACF and dNAP1 Reveals That Acetyl-CoA Is Required for Preinitiation Complex Assembly

Jiang¹,

Nordeen²,

Kadonaga³

2000

Journal of Biological Chemistry

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To investigate the role of chromatin structure in the regulation of transcription by RNA polymerase II, we developed a chromatin transcription system in which periodic nucleosome arrays are assembled with purified recombinant ATP-utilizing chromatin assembly and remodeling factor (ACF), purified recombinant nucleosome assembly protein 1 (dNAP1), purified native core histones, plasmid DNA, and ATP. With this chromatin, we observed robust activation of transcription with three different transcription factor sets (nuclear factor B p65 ؉ Sp1, estrogen receptor, and Gal4-VP16) added either before or after chromatin assembly. In fact, the efficiency of activated transcription from the ACF ؉ dNAP1-assembled chromatin was observed to be comparable with that from naked DNA templates or chromatin assembled with a crude Drosophila extract (S190). With ACF ؉ dNAP1-assembled chromatin, we found that transcriptional activation is dependent upon acetyl-CoA. This effect was not seen with naked DNA templates or with crude S190-assembled chromatin. We further determined that acetyl-CoA is required at the time of preinitiation complex assembly but not during assembly of the chromatin template. These findings suggest that there is at least one key acetylation event that is needed to assemble a functional transcription preinitiation complex with a chromatin template.The regulation of gene expression at the level of transcription is a key control point for many cellular processes. In eukaryotes, the transcription of protein-coding genes by RNA polymerase II occurs in the milieu of chromatin and involves the covalent and noncovalent modification of nucleosomes (for reviews, see Refs. 1-13). There are, for instance, enzymes that modify histones by acetylation/deacetylation, phosphorylation, methylation, ubiquitination, or ADP-ribosylation. In addition, ATP-dependent proteins, termed chromatin remodeling factors, alter histone-DNA contacts and catalyze nucleosome mobility. It will thus be important to understand the relation between chromatin structure and the activity of each of the tens of thousands of genes in an organism.To investigate the role of chromatin in transcriptional regulation, it is possible to use biochemical systems for the analysis of in vitro reconstituted chromatin. To this end, we previously developed a chromatin transcription system that is based on a crude chromatin assembly extract termed the S190 extract, which is derived from Drosophila embryos (14, 15). This S190-based chromatin transcription system has been used for the analysis of many transcription factors.Although the S190 extract has been a reliable source of chromatin assembly activity, we have recently achieved the ATP-dependent assembly of chromatin with purified, recombinant chromatin assembly factors (16). In these reactions, periodic nucleosome arrays are assembled with purified recombinant ACF, 1 purified recombinant dNAP1, purified native core histones, plasmid DNA, and ATP. dNAP1 functions stoichiometrically as a core histone chaperone, whereas A...

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Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro.

Cited by 177 publications

References 29 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.

Conservation of transcriptional activation functions of the NF-kappa B p50 and p65 subunits in mammalian cells and Saccharomyces cerevisiae.

Transcriptional Analysis of Chromatin Assembled with Purified ACF and dNAP1 Reveals That Acetyl-CoA Is Required for Preinitiation Complex Assembly

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