Mutational analysis of the DNA-binding domain of yeast heat shock transcription factor

Hubl, S; Owens, Julia C.; Nelson, Hillary C. M.

doi:10.1038/nsb0994-615

Cited by 31 publications

(36 citation statements)

References 29 publications

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“…2); mutation of this residue to alanine caused the protein to be proteolyzed (data not shown). Another residue, Trp 194 , lies in a hydrophobic pocket and is likely involved in inter-or intramolecular contacts (25). Given the location of the remaining four within or located near helix 3 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Experimental Rationale-In a previous study, we had used alanine-scanning mutagenesis, in combination with structural studies on the DNA-binding domain, to analyze systematically the surface residues of the HSF DNA-binding domain (25). The assay depended on the requirement of a functional HSF for yeast viability (3).…”

Section: Resultsmentioning

confidence: 99%

“…Plasmids and Yeast Strains-The DNA-binding domain mutants used for yeast expression were all constructed from pHN1002, a CEN/ AR4S/TRP1 yeast shuttle vector carrying a full-length copy of the S. cerevisiae HSF1 gene expressed from its own promoter (25). Construction of the full-length versions of the alanine-scanning mutants has been described (25).…”

Section: Methodsmentioning

confidence: 99%

“…Construction of the full-length versions of the alanine-scanning mutants has been described (25). C-terminal truncations of specific alaninesubstitutions were based on pHN2256, a version of pHN1002 that includes the first 583 residues of HSF followed by valine and asparagine as cloning artifacts and then a stop codon (gift of E. E. Powers).…”

Section: Methodsmentioning

confidence: 99%

“…In order to explore further the role of the DNA-binding domain in regulating transcriptional activity, we have studied the transcriptional activity of a series of alanine-scanning mutants that cover the surface of the DNA-binding domain (12,25). The majority of the surface residue mutations cause a change in transcriptional activity compared with wild-type HSF.…”

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confidence: 99%

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The DNA-binding Domain of Yeast Heat Shock Transcription Factor Independently Regulates Both the N- and C-terminal Activation Domains

Bulman¹,

Hubl²,

Nelson³

2001

Journal of Biological Chemistry

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The expression of heat shock proteins in response to cellular stresses is dependent on the activity of the heat shock transcription factor (HSF). In yeast, HSF is constitutively bound to DNA; however, the mitigation of negative regulation in response to stress dramatically increases transcriptional activity. Through alaninescanning mutagenesis of the surface residues of the DNA-binding domain, we have identified a large number of mutants with increased transcriptional activity. Six of the strongest mutations were selected for detailed study. Our studies suggest that the DNA-binding domain is involved in the negative regulation of both the Nterminal and C-terminal activation domains of HSF. These mutations do not significantly affect DNA binding. Circular dichroism analysis suggests that a subset of the mutants may have altered secondary structure, whereas a different subset has decreased thermal stability. Our findings suggest that the regulation of HSF transcriptional activity (under both constitutive and stressed conditions) may be partially dependent on the local topology of the DNA-binding domain. In addition, the DNA-binding domain may mediate key interactions with ancillary factors and/or other intramolecular regulatory regions in order to modulate the complex regulation of HSF's transcriptional activity.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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The DNA-binding Domain of Yeast Heat Shock Transcription Factor Independently Regulates Both the N- and C-terminal Activation Domains

Bulman¹,

Hubl²,

Nelson³

2001

Journal of Biological Chemistry

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Solution structure of the ETS domain from murine Ets-1: a winged helix-turn-helix DNA binding motif.

et al. 1996

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Ets‐1 is the prototypic member of the ets family of transcription factors. This family is characterized by the conserved ETS domain that mediates specific DNA binding. Using NMR methods, we have determined the structure of a fragment of murine Ets‐1 composed of the 85 residue ETS domain and a 25 amino acid extension that ends at its native C‐terminus. The ETS domain folds into a helix‐turn‐helix motif on a four‐stranded anti‐parallel beta‐sheet scaffold. This structure places Ets‐1 in the winged helix‐turn‐helix (wHTH) family of DNA binding proteins and provides a model for interpreting the sequence conservation of the ETS domain and the specific interaction of Ets‐1 with DNA. The C‐terminal sequence of Ets‐1, which is mutated in the v‐Ets oncoprotein, forms an alpha‐helix that packs anti‐parallel to the N‐terminal helix of the ETS domain. In this position, the C‐terminal helix is poised to interact directly with an N‐terminal inhibitory region in Ets‐1 as well as the wHTH motif. This explains structurally the concerted role of residues flanking the ETS domain in the intramolecular inhibition of Ets‐1 DNA binding.

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Protonation Behavior of Histidine during HSF1 Activation by Physiological Acidification

Lü

Park²

2015

J of Cellular Biochemistry

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The expression of eukaryotic molecular chaperones (heat shock proteins, HSPs) is triggered in response to a wide range of environmental stresses, including: heat shock, hydrogen peroxide, heavy metal, low-pH, or virus infection. Biochemical and genetic studies have clearly shown the fundamental roles of heat shock factor 1 (HSF1) in stress-inducible HSP gene expression, resistance to stress-induced cell death, carcinogenesis, and other biological phenomena. Previous studies show that acidic pH changes within the physiological range directly activate the HSF1 function in vitro. However, the detailed mechanism is unclear. Though computational pKa-predications of the amino acid side-chain, acidic-pH induced protonation of a histidine residue was found to be most-likely involved in this process. The histidine 83 (His83) residue, which could be protonated by mild decrease in pH, causes mild acidic-induced HSF1 activation (including in-vitro trimerization, DNA binding, in-vivo nuclear accumulation, and HSPs expression). His83, which is located in the loop region of the HSF1 DNA binding domain, was suggested to enhance the intermolecular force with Arginine 79, which helps HSF1 form a DNA-binding competent. Therefore, low-pH-induced activation of HSF1 by the protonation of histidine can help us better to understand the HSF1 mechanism and develop more therapeutic applications (particularly in cancer therapy). J. Cell. Biochem. 116: 977-984, 2015. © 2015 Wiley Periodicals, Inc.

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Mutational analysis of the DNA-binding domain of yeast heat shock transcription factor

Cited by 31 publications

References 29 publications

The DNA-binding Domain of Yeast Heat Shock Transcription Factor Independently Regulates Both the N- and C-terminal Activation Domains

The DNA-binding Domain of Yeast Heat Shock Transcription Factor Independently Regulates Both the N- and C-terminal Activation Domains

Solution structure of the ETS domain from murine Ets-1: a winged helix-turn-helix DNA binding motif.

Protonation Behavior of Histidine during HSF1 Activation by Physiological Acidification

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