Both randomized oligonucleotide cassette mutagenesis and site-directed mutagenesis have been used in combination with a yeast genetic screen to identify critical residues in the DNA-binding domain of heat shock transcription factor from Saccharomyces cerevisiae. Most of the surface residues in this highly conserved domain can be changed to alanine with no observable effect on function. Of nine critical residues identified in this screen, five are within helix α3, previously designated as the probable DNA recognition helix in the crystal structure of the Kluyveromyces lactis protein. The other four residues may be involved in DNA-binding or protein–protein interactions.
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Hubl, S., Owens, J. & Nelson, H. Mutational analysis of the DNA-binding domain of yeast heat shock transcription factor. Nat Struct Mol Biol 1, 615–620 (1994). https://doi.org/10.1038/nsb0994-615
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