Minimum length of a sequence-specific DNA binding peptide

Abstract

NMR experiments show that a stable complex can be formed between a 14-base-pair oligonucleotide and a disulfide-bonded dimer of a peptide containing 27 residues of the basic region of the yeast transcriptional activator GCN4; the complex is in slow exchange on the NMR time scale. In contrast, a nonspecific complex is in fast exchange on the NMR time scale. DNase I footprinting experiments show that dimers of peptides containing as few as 20 residues of GCN4 bind DNA with sequence specificity similar to that of the intact protein. Circular dichroism experiments suggest that specific binding involves only 15 residues, corresponding to residues 231-245 of GCN4, in an alpha-helical conformation. These results limit substantially the region of GCN4 involved in sequence-specific DNA contacts and provide a uniquely simple model for studying protein-DNA interactions in detail.

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

“…Particularly relevant are those based on dimeric bZIP basic regions, in which the natural C/C-terminal leucine zipper is replaced by artificial dimerizers [14][15][16][17] . Some of these designs have gone even further and incorporate switchable elements that allow conditional off/on DNA binding by the application of external stimuli such as light or metal ions [18][19][20][21][22] .…”

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

“…[11][12][13][14][15] For example, a study by Kim and co-workers, compared a library of peptide dimers (of different lengths) derived from the basic domain of the GCN4 transcription factor. These peptides all bore a GGC motif toward the C terminus to allow dimerisation through thiol oxidation.…”

“…Both DNase I footprinting and circular dichroism (CD) melting experiments revealed that the 15 amino acid sequence KRARNTEAARRSRAR contains the necessary conserved residues required for the formation of specific contacts with the DNA target site CRE, whereas binding to AP1 required additional conserved residues. [13] The linker region LQRMKQL, separating the basic and leucine zipper domains and which allows for the formation of an uninterrupted a-helix, [36] has been reported to be important for thermal stability on binding to both CRE and AP1 DNA. [11,13] Based on this literature our initial design, Ac-ALKRARN-TEAARRSRARKLQRMKQLGGCG-NH 2 , included both the basic domain and the flexible linker region so as to achieve strong and selective DNA binding.…”

“…[13] The linker region LQRMKQL, separating the basic and leucine zipper domains and which allows for the formation of an uninterrupted a-helix, [36] has been reported to be important for thermal stability on binding to both CRE and AP1 DNA. [11,13] Based on this literature our initial design, Ac-ALKRARN-TEAARRSRARKLQRMKQLGGCG-NH 2 , included both the basic domain and the flexible linker region so as to achieve strong and selective DNA binding. However, DNA binding was found to be sufficiently strong so that both the bipyridine and terpyridine peptide dimers bound to DNA in the absence of any metal ions (confirmed by gel electrophoresis), and the addition of Cu II or Zn II did not alter DNA binding.…”

“…[43] However, peptides derived from the basic domain of GCN4 have been shown to display sequence-specific folding into a-helices in the presence of DNA containing a GCN4 target site (such as CRE). [13] Therefore, CD, and specifically the ellipticity at 208 and 222 nm, can be used to infer DNA binding. This is particularly important as we were unable to obtain band retardation for any of the GCN4bd peptide dimer DNA complexes in gel electrophoresis band-shift assays in either the absence or presence of metal ions, even at CD relevant concentrations.…”