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. 1994 Jun 25;22(12):2198–2208. doi: 10.1093/nar/22.12.2198

Creating new DNA binding specificities in the yeast transcriptional activator GCN4 by combining selected amino acid substitutions.

M Suckow 1, A Madan 1, B Kisters-Woike 1, B von Wilcken-Bergmann 1, B Müller-Hill 1
PMCID: PMC523674  PMID: 8036145

Abstract

The specificity of the GCN4/DNA complex is mediated by a complicated network of interactions between the basic regions of both GCN4 monomers and their target halfsites. According to X-ray analyses (1, 2) one particular thymine of the target sequence is recognized by serine -11 and alanine -15 (we define the leucine in the first d-position of the heptad repeats as +1). We replaced serine -11 or alanine -15 with all other amino acids and analysed the DNA binding properties of the resulting stable GCN4 derivatives by electrophoretic mobility shift assays. Among these, mutants with tryptophan in position -11, or glutamic acid and glutamine in position -15, differ significantly from GCN4 in their DNA binding specificities. We then constructed selected double mutants, which differ from GCN4 in positions -11, -15 or -14 (3) of the basic region. The double mutants with tryptophan in position -11 and asparagine or serine in position -14 show drastically altered DNA binding specificities, presumably due to additive effects.

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Selected References

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