Creating new DNA binding specificities in the yeast transcriptional activator GCN4 by combining selected amino acid substitutions.
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.
Suckow
M
M
Institut für Genetik, Universität zu Köln, Germany.
Madan
A
A
Kisters-Woike
B
B
von Wilcken-Bergmann
B
B
Müller-Hill
B
B
D001483:Base Sequence; D003196:Computer Graphics; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D011494:Protein Kinases