GCN4 Binds with High Affinity to DNA Sequences Containing a Single Consensus Half-Site

Hollenbeck, Jessica J.; Oakley, Martha G.

doi:10.1021/bi992705n

Cited by 53 publications

(66 citation statements)

References 83 publications

(176 reference statements)

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“…This is in agreement with measurements by Hollenbeck and Oakley, who measured a 10-fold decrease in half-site binding (38). In related studies, other labs have found that monomeric and dimeric GCN4 bZIP bind similarly and strongly to their target half and full sites (41)(42)(43).…”

Section: Resultssupporting

confidence: 92%

“…For example, little or no induction of helicity in bZIP derivatives upon addition of nonspecific DNA has been observed in some cases (44,45). However, Hollenbeck and Oakley found that their nonspecific DNA control induced comparable helicity in their GCN4 bZIP derivative as did the native AP-1 and CRE sites (38). Regardless of strength of thermodynamic binding affinity, all of the sites that we examined by CD can preorganize R-helical structure, and there appears to be no clear relationship between binding affinities and structural stabilization.…”

Section: Resultsmentioning

confidence: 99%

“…As depicted in Figure 6, GCN4 is capable of binding to specific DNA targets utilizing three different binding modes: dimeric binding observed when GCN4 binds its canonical full sites AP-1 and CRE, dimeric binding to its canonical DNA half site TGAC in which one monomer binds specifically to the canonical half site and the other monomer binds to DNA nonspecifically (38), and monomeric binding to a single canonical half site (41,49). The wt bZIP is capable of selective dimeric binding to noncognate DNA full sites ( Figure 6A) (17).…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

The GCN4 bZIP Targets Noncognate Gene Regulatory Sequences: Quantitative Investigation of Binding at Full and Half Sites

2007

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: We previously reported that a basic region/leucine zipper (bZIP) protein, a hybrid of the GCN4 basic region and C/EBP leucine zipper, not only recognizes cognate target sites AP-1 (5′-TGACTCA-3′) and cAMP-response element (CRE) (5′-TGACGTCA-3′) but also binds selectively to noncognate DNA sites: C/EBP (CCAAT/enhancer binding protein, 5′-TTGCGCAA), XRE1 (xenobiotic response element, 5′-TTGCGTGA), HRE (HIF response element, 5′-GCACGTAG), and E-box (5′-CACGTG). In this work, we used electrophoretic mobility shift assay (EMSA) and circular dichroism (CD) for more extensive characterization of the binding of wt bZIP dimer to noncognate sites as well as full-and half-site derivatives, and we examined changes in flanking sequences. Quantitative EMSA titrations were used to measure dissociation constants of this hybrid, wt bZIP, to DNA duplexes: Full-site binding affinities gradually decrease from cognate sites AP-1 and CRE with K d values of 13 and 12 nM, respectively, to noncognate sites with K d values of 120 nM to low µM. DNA-binding selectivity at half sites is maintained; however, half-site binding affinities sharply decrease from the cognate half site (K d ) 84 nM) to noncognate half sites (all K d values > 2 µM). CD shows that comparable levels of R-helical structure are induced in wt bZIP upon binding to cognate AP-1 or noncognate sites. Thus, noncognate sites may contribute to preorganization of stable protein structure before binding target DNA sites. This work demonstrates that the bZIP scaffold may be a powerful tool in the design of small, R-helical proteins with desired DNA recognition properties.In order to examine the relationship between protein structure and DNA-binding function, we exploit the protein R-helix, a structure used ubiquitously for sequence-selective DNA recognition and one that chemists have successfully manipulated in design and synthesis studies for many years (examples include refs 1-8). GCN4 is a dimeric transcriptional regulator that governs histidine biosynthesis in yeast under conditions of amino acid starvation (9). Crystal structures of the basic region/leucine zipper (bZIP) 1 domain of GCN4 bound to the AP-1 site (5′-TGACTCA) (10) and cAMP-response element (CRE) site (5′-TGACGTCA) (11, 12) and the Jun-Fos bZIP heterodimer bound to show that a continuous R-helix provides the basic region interface for binding to specific DNA sites, as well as the leucine zipper coiled coil dimerization structure. The fulllength GCN4 monomer is 281 amino acids, and the bZIP comprises a dimer of ∼60 residue monomers. We previously generated a series of minimalist bZIP proteins comprising alanine-rich variants of the GCN4 basic region fused to the C/EBP leucine zipper: The wt bZIP comprises the native GCN4 basic region. McKnight and co-workers showed that exchanging basic regions and leucine zippers between GCN4 and C/EBP resulted in functional proteins that targeted cognate DNA sites (14), and likewise, we found our wt bZIP hybrid to be functionally equivalent to the GCN4 bZIP (15,16).

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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The GCN4 bZIP Targets Noncognate Gene Regulatory Sequences: Quantitative Investigation of Binding at Full and Half Sites

2007

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“…Previous studies have demonstrated that homodimeric peptides based on GCN4 which contain both the conserved basic and zipper domains, display similar binding affinities for CRE, AP1 and half-CRE DNA. [59] A slight increase in the helicity of (GCN4bd) 2 and bipy-(GCN4bd) 2 is observed in their CD spectra recorded in the presence of either AP1 or half-CRE DNA, similar to that in the presence of CRE DNA (but in the absence of metal ions), and to previous reports ( Figure 7 A and B).…”

Section: Dna Sequence Selectivitysupporting

confidence: 88%

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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“…3b, lanes 1-3, and 7-9). These bands with the non-consensus dsDNAs are more diffuse than those observed with the direct DNA, and most probably arise from a relatively loose complex in which one of the basic regions is interacting specifically in the major groove of the consensus site while the other makes nonspecific contacts 38 . Curiously, the control disulphide (3b) 2 SS, in which the terpyridine was replaced by an inert chromophore, did not elicit such interactions with the non-consensus DNAs (Fig.…”

Section: Resultsmentioning

confidence: 88%

Stimuli-responsive selection of target DNA sequences by synthetic bZIP peptides

Mosquera¹,

Jiménez-Balsa²,

Dodero³

et al. 2013

Nat Commun

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One of the strategies used by nature to regulate gene expression relies on the stimulicontrolled combination of DNA-binding proteins. This in turn determines the target-binding site within the genome, and thereby whether a particular gene is activated or repressed. Here we demonstrate how a designed basic region leucine zipper-based peptide can be directed towards two different DNA sequences depending on its dimerization arrangement. While the monomeric peptide is non-functional, a C-terminal metallo-dimer recognizes the natural ATF/CREB-binding site (5 0 -ATGA cg TCAT-3 0 ), and a N-terminal disulphide dimer binds preferentially to the swapped sequence (5 0 -TCAT cg ATGA-3 0 ). As the dimerization mode can be efficiently controlled by appropriate external reagents, it is possible to reversibly drive the peptide to either DNA site in response to such specific inputs. This represents the first example of a designed molecule that can bind to more than one specific DNA sequence depending on changes in its environment.

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GCN4 Binds with High Affinity to DNA Sequences Containing a Single Consensus Half-Site

Cited by 53 publications

References 83 publications

The GCN4 bZIP Targets Noncognate Gene Regulatory Sequences: Quantitative Investigation of Binding at Full and Half Sites

The GCN4 bZIP Targets Noncognate Gene Regulatory Sequences: Quantitative Investigation of Binding at Full and Half Sites

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

Stimuli-responsive selection of target DNA sequences by synthetic bZIP peptides

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