Genetic evidence that an activation domain of GAL4 does not require acidity and may form a β sheet

Leuther, Kerstin K.; Salmeron, John M.; Johnston, Stephen Albert

doi:10.1016/0092-8674(93)90076-3

Cited by 140 publications

(109 citation statements)

References 68 publications

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“…Because the TAD-target binding almost entirely relies on hydrophobic steric fit (7-9, 16, 26), not only the N-terminal subdomain containing the strongly amphipathic helix but also the C-terminal subdomain containing weakly amphipathic turns is expected to be able to bind to a same target protein. The fact that binding affinity of a p53 TAD fragment (residues 1-57) to mdm2 is higher by ϳ50% than that of a shorter fragment (residues [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] is consistent with such a prediction (7). The N-terminal subdomain, having an amphipathic helix, should bind more efficiently and produce stronger activity than the C-terminal subdomain that contains a nascent turn (note that neither subdomain contains the turn I formed by residues Met 40 -Met 44 due to the particular way the full-length p53 TAD is split).…”

Section: Resultsmentioning

confidence: 99%

“…Even though the minimal activation domain from the human glucocorticoid receptor was shown to contain multiple helices, this observation was possible only in the presence of a strong helix-promoting solvent (23). The presence of some secondary structures was noted by CD measurements in the activation domains of GCN4 and GAL4 in a hydrophobic solvent, but the dominant form was controversially found to be antiparallel ␤-sheet rather than the putative ␣-helix (24,25). The results of NMR studies on short TAD fragments are also controversial.…”

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confidence: 99%

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Local Structural Elements in the Mostly Unstructured Transcriptional Activation Domain of Human p53

Lee

Mok

Muhandiram

et al. 2000

Journal of Biological Chemistry

331

336

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DNA transcription is initiated by a small regulatory region of transactivators known as the transactivation domain. In contrast to the rapid progress made on the functional aspect of this promiscuous domain, its structural feature is still poorly characterized. Here, our multidimensional NMR study reveals that an unbound fulllength p53 transactivation domain, although similar to the recently discovered group of loosely folded proteins in that it does not have tertiary structure, is nevertheless populated by an amphipathic helix and two nascent turns.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Local Structural Elements in the Mostly Unstructured Transcriptional Activation Domain of Human p53

Lee

Mok

Muhandiram

et al. 2000

Journal of Biological Chemistry

331

336

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“…Indeed, the deletion analysis (Figure 1) demonstrates that a region which is remarkably rich in glutamines (34% glutamines between positions 470 and 560) contains no activation potential in the yeast system. Furthermore, the K1HSF activator is only slightly acidic having a calculated net charge of -5 and may therefore be atypical for yeast activators in which acidity is a characteristic, though probably not essential, feature (Giniger and Leuther et al, 1993). In an attempt to clarify the importance of the negative charge for this activator we introduced mutations which alter the charge to +3; this causes an -6-fold reduction, although not a complete loss, of activity.…”

Section: Discussionmentioning

confidence: 99%

Identification of the C-terminal activator domain in yeast heat shock factor: independent control of transient and sustained transcriptional activity.

et al. 1993

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In yeast, heat shock factor (HSF) is a trimer that binds DNA constitutively but only supports high levels of transcription upon heat shock. The C-terminal regions of HSF from Saccharomyces cerevisiae and Kluyveromyces lactis are unconserved yet both contain strong transactivators which are correctly regulated when substituted for each other. We have performed high resolution mapping of these activator domains which shows that in K.lacts HSF (KIHSF) activity can be located to a confined short domain, while in S.cerevisiae HSF (ScHSF) two separate regions are required for full activity. Alignment of the activator domains reveals similarity, as both overlap potential leucine zipper motifs (zipper C) with a distribution of hydrophobic residues sinilar to two highly conserved N-terminal domains which mediate HSF trimerization (zippers A and B). In higher eukaryotes a C-terminal leucine zipper is required to maintain HSF in a monomeric and non DNA-binding state under normal conditions and we therefore address the regulatory roles of the three leucine zipper motifs in KIHSF. Whilst the longest and most N-terminal of the trimer region zippers, A, is dispensable for regulation, mutation of a single leucine in zipper B makes HSF constitutively active. In contrast to the situation in higher eukaryotes disruption of zipper C has no observable regulatory effect and therefore, although an intramolecular contact between zippers B and C cannot be ruled out, such contact is not required for restraining the C-terminal activator domain. We furthermore fimd that deletions which abolish activator potential of the C-terminus render the host strain temperature sensitive.However, deletion of a double proline-glycine motif in the activator, whilst leaving HSF unable to respond to heat shock, does not cause temperature sensitivity. This result demonstrates that independent mechanisms control the transient and sustained activities of HSF.

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“…Yeast Culturing and ␣-Galatosidase Assays-Yeast culture conditions and ␣-galactosidase assays were performed as described (14). Protein concentrations of the yeast extracts were determined using the BCA protein assay reagent (Pierce) and bovine serum albumin to generate the standard curve.…”

Section: Methodsmentioning

confidence: 99%

Alterations in the GAL4 DNA-binding Domain Can Affect Transcriptional Activation Independent of DNA Binding

Corton

Granell

Johnston

1998

Journal of Biological Chemistry

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The GAL4 protein belongs to a large class of fungal transcriptional activator proteins encoding within their DNA-binding domains (DBD) six cysteines that coordinate two atoms of zinc (the Zn 2 Cys 6 domain). In an effort to characterize the interactions between the Zn 2 Cys 6 class transcriptional activator proteins and their DNAbinding sites, we have replaced in the full-length GAL4 protein small regions of the Zn 2 Cys 6 domain with the analogous regions of another Zn 2 Cys 6 protein called PPR1 an activator of pyrimidine biosynthetic genes. Alterations between the first and third cysteines abolished binding to GAL4 (upstream activation sequence of GAL (UAS G )) or PPR1 (upstream acitvation sequence of UAS) DNA-binding sites and severely reduced transcriptional activation in yeast. In contrast, alterations between the third and fourth cysteines had only minor effects on binding to UAS G but led to substantial decreases in activation in both yeast and a mammalian cell line. In the crystal structure of the GAL4 DBD-UAS G complex (Marmorstein, R., Carey, M., Ptashne, M., and Harrison, S. C. (1992) Nature 356, 408 -414), this region is facing away from the DNA, making it likely that there exists within the GAL4 DBD an accessible domain important in activation.The GAL4 protein is an 881-amino acid transcriptional activator of the GAL and MEL1 genes in Saccharomyces cerevisiae. The mechanism by which GAL4 activates transcription is conserved because GAL4 also activates transcription in plant, Drosophila, and mammalian cells if GAL4 DNA-binding sites (UAS G ) 1 are present in the promoter of appropriate reporter genes (reviewed in Ref. 1). GAL4 is a member of a large family of fungal transcriptional activators that contain within their DNA-binding domains a conserved cysteine-rich region encoding six cysteine residues that coordinate two atoms of zinc (the Zn 2 Cys 6 domain). The DNA recognition sequences to which these activators bind have been identified and all have in common a CGG or related triplet in each of the two symmetrically opposed half-sites (2-4). The sequences between the triplets as well as the spacing of the triplet sequence are highly variable, indicating that these variables may in large part determine specific recognition. Based on the extent of homology between the Zn 2 Cys 6 activators, it was hypothesized that the Cys-rich region performs a function common to all members of this class of proteins and the region immediately adjacent, which encodes divergent sequences, determines DNA binding specificity (5). We previously demonstrated that this model is essentially correct (6). All but one (Lys 23 ) of the 28 amino acids in the Zn 2 Cys 6 region of GAL4 can be replaced by the analogous sequences of PPR1, an activator of pyrimidine biosynthetic genes (7), without changing DNA binding specificity. In contrast, replacing the 14 amino acids immediately adjacent to the Zn 2 Cys 6 region resulted in switching the DNA binding specificity to that of PPR1. Similar results were obtained when hybrids of L...

show abstract

Genetic evidence that an activation domain of GAL4 does not require acidity and may form a β sheet

Cited by 140 publications

References 68 publications

Local Structural Elements in the Mostly Unstructured Transcriptional Activation Domain of Human p53

Local Structural Elements in the Mostly Unstructured Transcriptional Activation Domain of Human p53

Identification of the C-terminal activator domain in yeast heat shock factor: independent control of transient and sustained transcriptional activity.

Alterations in the GAL4 DNA-binding Domain Can Affect Transcriptional Activation Independent of DNA Binding

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