FIG. 5.

Specific hydrophobic amino acids are essential for transcriptional activation by the Hap4 aa 359 to 476 fragment. (A) Amino acid sequence of Hap4(359-476) shown in single-letter code. Underlined positions indicate sites of serine substitutions; circled numbers identify the seven multiple-substitution mutants. (B) LexA-Hap4(359-476) fusion proteins, with or without the indicated substitutions, were expressed in L40 and assayed for activation of the integrated lacZ reporter gene as described for Fig. 2. The histograms indicate means (with standard deviations) of β-galactosidase activity in yeast cell extracts from three parallel cultures. (C) Immunoblot of cell lysates expressing LexA-Hap4 fusion proteins (wild-type or substitution mutants 1 through 7) probed with a panel of monoclonal antibodies recognizing the LexA DNA-binding domain. Mutant proteins 1 and 2 consistently showed slightly retarded mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.