Replacement of invariant bzip residuces within the basic region of the yeast transcriptional activator GCN4 can Change its DNA binding specificity

Suckow, Manfred; Schwamborn, Klaus; Kisters‐Woike, Brigitte; Wilcken‐Bergmann, Brigitte von; Müller‐Hill, Benno

doi:10.1093/nar/22.21.4395

Cited by 46 publications

(49 citation statements)

References 28 publications

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“…Of the 38 proteins, 19 closely match the canonical bZIP definition, including having an Asn at the location corresponding to residue 235 of S. cerevisiae GCN4. This site was described previously as "invariant" due to its conservation in bZIPs from mammals, plants, and fungi and was shown by crystallographic studies to interact with DNA (5,40,41). The 19 remaining proteins had substitutions at position 235, with 15 containing Cys, three containing Val, and one containing Tyr.…”

Section: Resultsmentioning

confidence: 78%

“…The diversification of bZIPs in oomycetes compared to those of model animals, fungi, and plants demonstrates the value of exploring diverse taxa. Mutation studies in model species indicate that exchanging Asn for other amino acids does not block DNA binding but alters the DNA-binding site (4)(5)(6). No code relating the sequence of the DNA-binding domain to its preferred target site is known, and even the highly conserved Asn is known to bind DNA in alternative configurations in different bZIPs (40,55).…”

Section: Discussionmentioning

confidence: 99%

“…Prior studies identified two sites in the DNA-binding domain that are invariant between bZIPs: an Asn at the position corresponding to residue 235 of the well-studied GCN4 transcription factor of Saccharomyces cerevisiae and Arg at position 243 (1)(2)(3). Mutations that alter these do not eliminate the ability of a bZIP to bind DNA but change the preferred target sequence (4)(5)(6).…”

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confidence: 99%

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bZIP Transcription Factors in the Oomycete Phytophthora infestans with Novel DNA-Binding Domains Are Involved in Defense against Oxidative Stress

2013

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Transcription factors of the basic leucine zipper (bZIP) family control development and stress responses in eukaryotes. To date, only one bZIP has been described in any oomycete; oomycetes are members of the stramenopile kingdom. In this study, we describe the identification of 38 bZIPs from the Phytophthora infestans genome. Half contain novel substitutions in the DNAbinding domain at a site that in other eukaryotes is reported to always be Asn. Interspecific comparisons indicated that the novel substitutions (usually Cys, but also Val and Tyr) arose after oomycetes diverged from other stramenopiles. About two-thirds of P. infestans bZIPs show dynamic changes in mRNA levels during the life cycle, with many of the genes being upregulated in sporangia, zoospores, or germinated zoospore cysts. One bZIP with the novel Cys substitution was shown to reside in the nucleus throughout growth and development. Using stable gene silencing, the functions of eight bZIPs with the Cys substitution were tested. All but one were found to play roles in protecting P. infestans from hydrogen peroxide-induced injury, and it is proposed that the novel Cys substitution serves as a redox sensor. A ninth bZIP lacking the novel Asn-to-Cys substitution, but having Cys nearby, was also shown through silencing to contribute to defense against peroxide. Little effect on asexual development, plant pathogenesis, or resistance to osmotic stress was observed in transformants silenced for any of the nine bZIPs.

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Section: Resultsmentioning

confidence: 78%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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bZIP Transcription Factors in the Oomycete Phytophthora infestans with Novel DNA-Binding Domains Are Involved in Defense against Oxidative Stress

2013

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“…An aliquot of 2 µL of a serial dilution of calf thymus DNA (CT DNA) was added to 8 µL of binding buffer [1.4 mM KH 2 PO 4 , 4.3 mM Na 2 HPO 4 , 2.7 mM KCl, 137 mM NaCl (pH 7.4), 1 mM EDTA, 0.1% NP-40, 0.4 mg‚mL -1 BSA, and 5% glycerol] containing a fixed concentration of peptide sufficient for a 50-80% mobility shift in the absence of competitor DNA and e80 pM 32 P-labeled hsCRE 25 DNA. The binding reactions were equilibrated for 1 h at 25°C, applied to a preequilibrated polyacrylamide gel, and electrophoresed as described above.…”

Section: Competition Electrophoretic Mobility Shiftmentioning

confidence: 99%

Relationship between Folding and Function in a Sequence-Specific Miniature DNA-Binding Protein

Yang

Schepartz

2005

Biochemistry

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Previously, we have described a miniature protein-based approach to the design of molecules that bind DNA or protein surfaces with high affinity and specificity. In this approach, the small, wellfolded protein avian pancreatic polypeptide acts as a scaffold to present and stabilize an R-helical or PPII-helical recognition epitope. The first miniature protein designed in this way, a molecule called p007, presents the R-helical recognition epitope found on the bZIP protein GCN4 and binds DNA with nanomolar affinity and exceptional specificity. In this work we use alanine-scanning mutagenesis to explore the contributions of 29 p007 residues to DNA affinity, specificity, and secondary structure. Virtually every residue within the p007 R-helix, and most residues within the p007 PPII helix, contribute to both DNA affinity and specificity. These residues include those introduced to make specific and nonspecific DNA contacts, as well as those that complete the miniature protein core. Moreover, there exists a direct correlation between the affinity of a p007 variant for specific DNA and the ability of that variant to select for specific DNA over nonspecific DNA. Although we observe no correlation between R-helicity and affinity, we observe a limited correlation between R-helicity and sequence specificity that emphasizes the role of coupled binding/folding in the function of p007. Our results imply that formation of a highly evolved set of protein‚DNA contacts in the context of a well-packed hydrophobic core, and not the extent of intrinsic R-helical structure, is the primary determinant of p007 function.The development of general strategies for the selective recognition of macromolecular targets remains a major challenge for chemical biology and a fundamental postgenome goal. Molecules capable of tight and selective macromolecular recognition have utility in the validation of potential therapeutic targets and can, in certain cases, function as therapeutics in their own right (1-3). Our laboratory has developed a general approach toward the design of small, well-folded proteins that bind macromolecular targets with high affinity and selectivity (4-11). This approach is often referred to as protein grafting, and the molecules that result are called miniature proteins: miniature because they contain fewer than 40 amino acids and proteins because they often fold cooperatively. In a protein grafting experiment, those residues that comprise a natural R-helical or type II polyproline (PPII)-helical recognition epitope are introduced onto the solvent-exposed R-or PPII-helical face of the small yet stable protein avian pancreatic polypeptide (aPP) 1 (12, 13). Our laboratory has used this procedure, often in combination with directed evolution, to engineer miniature protein ligands with high affinity for a variety of targets including the duplex DNAs recognized by GCN4 and CREB (4, 5) and the Q50K engrailed homeodomain (7), the antiapoptotic proteins Bcl-X L (6) and Bcl-2 (10), the oncoprotein hDM2 (manuscript in prepara...

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“…We propose to test the applicability of our metal-ion regulation sites as artificial dimerisation units for GCN4, which represents a well-studied system with a plethora of mutagenesis and (minimal) sequence studies identifying features important for selective DNA binding. [28][29][30][31][32] This report therefore describes the synthesis and characterisation of miniature DNA binding proteins, based on the basic domain of GCN4, dimerised by metal chelating bipyridine and terpyridine units. The Cu II and Zn II binding properties are evaluated and how pyridine ring rotation and subsequent reorientation of the peptide substituents, regulates DNA binding is reported.…”

Section: Introductionmentioning

confidence: 99%

Metal‐Ion‐Regulated Miniature DNA‐Binding Proteins Based on GCN4 and Non‐native Regulation Sites

Oheix

Peacock

2014

Chemistry A European J

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The design of artificial peptide dimers containing polypyridine switching domains, for which metal-ion coordination is shown to regulate DNA binding, is reported. Short peptides, based on the basic domain of the GCN4 transcription factor (GCN4bd), dimerised with either 2,2'-bipyridine (bipy(GCN4bd) 2 ) or 2,2':6',2''-terpyridine (terpy(GCN4bd) 2 ) linker units, undergo a conformational rearrangement on Cu II and Zn II coordination. Depending on the linker substitution pattern, this is proposed to alter the relative alignment of the two peptide moieties, and in turn regulate DNA binding. Circular dichroism and UV-visible spectroscopy reveal that Cu II and Zn II coordination promotes binding to DNA containing the CRE target site, but to a differing and opposite degree for the two linkers, and that the metal-ion affinity for terpy(GCN4bd) 2 is enhanced in the presence of CRE DNA. Binding to DNA containing the shorter AP1 target site, which lacks a single nucleobase pair compared to CRE, as well as half-CRE, which contains only half of the CRE target site, was also investigated. Cu II and Zn II coordination to terpy(GCN4bd) 2 promotes binding to AP1 DNA, and to a lesser extent half-CRE DNA. Whereas, bipy(GCN4bd) 2 , for which interpeptide distances are largely independent of metal-ion coordination and less suitable for binding to these shorter sites, displays allosteric ineffective behaviour in these cases. These findings for the first time demonstrate that biomolecular recognition, and specifically sequence-selective DNA binding, can be controlled by metal-ion coordination to designed switching units, non-native regulation sites, in artificial biomolecules. We believe that in the future these could find a wide range of applications in biotechnology.

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Replacement of invariant bzip residuces within the basic region of the yeast transcriptional activator GCN4 can Change its DNA binding specificity

Cited by 46 publications

References 28 publications

bZIP Transcription Factors in the Oomycete Phytophthora infestans with Novel DNA-Binding Domains Are Involved in Defense against Oxidative Stress

bZIP Transcription Factors in the Oomycete Phytophthora infestans with Novel DNA-Binding Domains Are Involved in Defense against Oxidative Stress

Relationship between Folding and Function in a Sequence-Specific Miniature DNA-Binding Protein

Metal‐Ion‐Regulated Miniature DNA‐Binding Proteins Based on GCN4 and Non‐native Regulation Sites

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