Figure 4.—
Put3 binds to the promoter of MCH5 and deletion of the binding site reduces its expression. (A and B) A double-stranded, Cy5-labeled DNA probe covering the Put3-binding site of the MCH5 promoter was incubated with protein extracts from ammonium-grown cells. As indicated, these cells were either wild type, overexpressing PUT3 from a multicopy plasmid (2μ PUT3), or put3Δ mutants. The binding reactions were analyzed by nondenaturing polyacrylamid gel electrophoresis, followed by fluorescence scanning. In B, a fourfold excess of the unlabeled probe was present during the binding assay. The positions of the free probes and of the bands appearing in cells overexpressing PUT3 are marked with arrows. (C) A GFP reporter construct containing the Put3 binding site from the MCH5 promoter in a MEL1 minimal promoter was transformed into BY4742-based strains (solid bars), or into MB758-5B (wt), or an isogenic PUT3c strain (open bars). The cells were grown in media containing 0.2 mg/liter riboflavin for 6 hr and GFP fluorescence was determined as described in materials and methods. (D) The same strains used in C carrying a similar plasmid lacking the Put3 binding site were subjected to GFP reporter assays.