DNA recognition by GAL4: structure of a protein-DNA complex

Marmorstein, Ronen; Carey, Michael; Ptashne, Mark; Harrison, Stephen C.

doi:10.1038/356408a0

Cited by 647 publications

(563 citation statements)

References 59 publications

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“…The DNA targets of several zinc cluster proteins have been identified. In general, the genes responding to such factors have upstream activating sequences (UAS) consisting of two CGG triplets separated by a number of basepairs that appear specific for each zinc cluster protein, for example 11 and 6 for the well-characterized Gal4p-DNA and Ppr1p-DNA complexes respectively (Marmorstein et al, 1992;Marmorstein and Harrison, 1994). The CGG triplets are essential for binding, whereas the internal sequences appear less important, at least in vitro.…”

Section: Introductionmentioning

confidence: 99%

A nonameric core sequence is required upstream of the LYS genes of Saccharomyces cerevisiae for Lys14p‐mediated activation and apparent repression by lysine

Becker

Feller

Alami

et al. 1998

Molecular Microbiology

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SummaryThe expression of the structural genes for lysine (LYS) biosynthesis is controlled by a pathway-specific regulation mediated by the transcriptional activator Lys14 in the presence of ␣-aminoadipate semialdehyde, an intermediate of the pathway acting as a coinducer. Owing to end product inhibition of the first step of the pathway, excess lysine reduces the production of the co-inducer and causes apparent repression of the LYS genes. Analysis of LYS promoters and insertions within an heterologous reporter gene have allowed the characterization of an upstream activating element (UAS LYS ) able to confer Lys14-and ␣-aminoadipate semialdehyde-dependent activation as well as apparent repression by lysine to another yeast gene. This DNA motif is present as one or several copies in the promoters of at least six LYS genes. The consensus sequence derived from the comparison of the UAS LYS showing the highest activation capacities comprises the nonameric core sequence TCCRNYGGA. The RNY sequence of the 3 bp spacer as well as the presence of flanking AT-rich regions on both sides of the core sequence appear essential for optimal activation. Further evidence that this element is the target of Lys14p was provided by the demonstration that Lys14p binds to UAS LYS in vitro. The binding is independent of the presence of the co-inducer and is not affected by lysine. It depends on the integrity of the putative Zn(II) 2 Cys 6 binuclear cluster contained in the Lys14p.

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Section: Introductionmentioning

confidence: 99%

A nonameric core sequence is required upstream of the LYS genes of Saccharomyces cerevisiae for Lys14p‐mediated activation and apparent repression by lysine

Becker

Feller

Alami

et al. 1998

Molecular Microbiology

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“…Their ! )NA binding domains conform to a Zn(II)2-Cys6 cluster lo-, ated near the N-terminus [1,2], they have acidic-hydrophobic ranscriptional activation domains, usually located near the C~erminus [3][4][5], and some of them are modulated, i.e. changed ~rom an inactive to an active form by a small effector moleule.…”

Section: ~ Introductionmentioning

confidence: 99%

Regulation of transcription in mammalian cells by yeast Leu3p and externally supplied inducer

Guo

Kohlhaw

1996

FEBS Letters

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The Leu3 protein of yeast is a dual-function regulator, stimulating transcription when the inducer ¢z-isopropyimalate (tx-IPM) is present and suppressing transcription when 1he inducer is absent. Here we show that Leu3p retains both its positive and negative regulatory properties when expressed in mammalian cells or when added to a mammalian nuclear extract. ~-IPM stimulates reporter gene expression 15-20-fold, both in ivo and in vitro. shown to stimulate transcription not only in yeast, but also in mammalian cells, consistent with the idea that the apparatus involved in gene activation has been conserved among eukaryotes [14,15]. By contrast, modulation of yeast (or other lower eukaryotic) transactivators apparently has not been demonstrated outside of their native environment. In some instances, such as in the case of Gal4p, modulation across species would be difficult to achieve since additional protein factors are required for masking and unmasking of the activation domain, and possibly other functions [16,17]. Experiments with LAC9, a Gal4p-related transactivator from Kluyveromyces lactis, support this notion: to achieve masking of LAC9 in mammalian cells, it was necessary to co-express GAL80 [18]. Moreover, masking could not be reversed by galactose, presumably because factors required for the unmasking process are absent from mammalian cells. We report here that Leu3p adopts a masked conformation in mammalian cells on its own and that unmasking can be achieved simply by including the inducer c~-IPM in the culture medium. Masked Leu3p causes repression, unmasked Leu3p causes activation of gene expression. Materials and methods Plasmid constructionPlasmid pC3-LEU3 was constructed to express full-length Leu3p from the human cytomegalovirus (CMV) major intermediate early promoter in mammalian cells. A 3.1 kb fragment containing the LEU3 sequence from -45 to +3063 [19] was excised from plasmid pZRL [20] by complete digestion with EcoRl and partial digestion with HindlII. The fragment was separated on a 0.7% agarose gel and extracted with the QIAEX II gel extraction kit (QIAGEN Inc.). It was then ligated into the polylinker region of the expression vector pcDNA3 (Invitrogen) that had also been digested with EcoRI and HindlII. The plasmid carrying the luciferase (LUC) reporter gene was pGL3-LUC (Promega). To construct pGL3-UASL-LUC, four consecutive UASLEu 30-mers [10] were inserted at the SacI site of the polylinker region of pGL3-LUC. Transfection of mammalian cells and luciferase assaysMouse 30A5 preadipocytes [21], a cell line derived from 10Tl/2 fibroblasts and kindly provided by K.-H. Kim, were cultured to about 70% confluence, then transfected, incubated, and lysed according to published procedures [22]. Luciferase assays were performed by luminometry following the supplier's protocol (Promega, Publication TM033). Transfection efficiency was monitored by cotransfecting the cells with pRSV-lacZ [22] and measuring I~-galactosidase activities. Preparation of nuclear extracts and in vitro transcripti...

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“…The positions between these motives are thought to be much less important consistent with the DNA-bound GAL4-DBD crystal structure. [31][32][33] To evaluate GAL4 binding to putative new recognition sites of Mt1-5 in vitro, gel shift experiments were performed (Fig 2a and b). However, only three of the five oligonucleotide duplexes tested bind recombinant GAL4-protein in vitro.…”

Section: Luciferase and Caspase-3 Assaysmentioning

confidence: 99%

Tumor-specific adenoviral gene therapy: transcriptional repression of gene expression by utilizing p53-signal transduction pathways

et al. 2003

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Adenoviral gene expression that is repressed by p53 in nontransformed cells could provide a tumor-specific gene therapy approach for a large subset of tumors. Adenoviral infection in vivo induces stabilization of p53, which can be utilized for a strategy that includes p53-dependent expression of a transcriptional repressor and a target promoter ,which is highly susceptible for transcriptional repression. Therefore, we constructed different versions of CMV-promoters (CMV gal ) with binding sites for GAL4-DBD and investigated 11 GAL4-DBD fusion proteins to elucidate the most effective repressor domain to silence CMV gal activity.The transcriptional repressor GAL4-KRAB-A under control of a p53-dependent promoter facilitates strong CMV gal -mediated gene expression specifically in p53 mutant cells by a double-recombinant adenoviral vector (Ad-RGCdR). GAL4-KRAB-A mediates strong transcriptional repression of Ad-RGCdR in p53 wild-type cells, which could be further enhanced by preactivation of p53-signalling following low-dose chemotherapy prior to adenoviral infection. By utilizing p53 signalling involved in chemotherapy and adenoviral infection, more than 99% of Ad-RGCdR gene expression could be repressed in p53 wild-type cells. Controlled gene expression from CMV gal promoters by transcriptional repression utilizing functional p53 signalling thus provides a very effective tool for tumor-specific adenoviral gene therapy.

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DNA recognition by GAL4: structure of a protein-DNA complex

Cited by 647 publications

References 59 publications

A nonameric core sequence is required upstream of the LYS genes of Saccharomyces cerevisiae for Lys14p‐mediated activation and apparent repression by lysine

A nonameric core sequence is required upstream of the LYS genes of Saccharomyces cerevisiae for Lys14p‐mediated activation and apparent repression by lysine

Regulation of transcription in mammalian cells by yeast Leu3p and externally supplied inducer

Tumor-specific adenoviral gene therapy: transcriptional repression of gene expression by utilizing p53-signal transduction pathways

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