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Nucleic Acids Research, 1994, Vol. 22, No. 12 2198-2208
© 1994

MOLECULAR BIOLOGY

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

Manfred Suckow, Anup Madan+, Brigitte Kisters-Woike, Brigitte von Wilcken-Bergmann and Benno Müller-Hill*

Institut für Genetik der Universität zu Köln Weyertal 121, 50931 Köln, Germany

*To whom correspondence should be addressed

Received May 4, 1994. Accepted May 18, 1994.

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.

+Presem address: Chemical Physics Group of the Tata Institute of Fundamental Research, Homi Bhabha, Colaba, Bombay-4000 005, India


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