FIG 3.

Protein 4 mutants with reduced RNA-binding activity in the Y3H system. (A) Random linker insertion mutagenesis. Schematic presentation of Protein 4 ORF (black horizontal line) with randomly inserted linkers (arrows) positions, each disrupted lacZ activation in the Y3H system. A stack of two arrows represents insertions of two different linkers at the same position. An arrow with a pentagon represents two identical insertions and thus, may not represent two independent insertion events. Gray bars denote positions of arginine or lysine residues. Black bars mark the position of positive residues subjected to site-directed mutagenesis. “W” marks a region with multiple in-frame insertions and a deletion. (B) Effects of point mutations in Protein 4 on its RNA-binding activity. Indicated Protein 4 mutants were tested in the Y3H system for lacZ activation by quantitative β-galactosidase liquid assay. Each mutant and its cognate wt clone were tested in parallel for binding to 5′ Segment 7 hybrid RNA. The boxplot presents the average β-galactosidase activity (with error bars; n = 3 yeast colonies per protein-RNA pair). Significance between pairs of wt and mutant was calculated using Student’s t test (***, P ≤ 0.001; NS, not significant). (C) Expression of wt and Protein 4 mutants in the yeast. Protein extracts of yeast expressing wt and the indicated Protein 4 mutants were analyzed by Western blotting with antibodies against Protein 4 and actin.