Fig. 2: Multiple regions of the Puf3-IDR interact with Ccr4–Not.
From: Phosphorylation-dependent tuning of mRNA deadenylation rates
a,b, Relative deadenylation rates of Ccr4–Not with mutant Puf3 proteins, plotted as t1/2. Experiments were performed in triplicate (open circles) and averaged (line). In a, Puf3 residues 53–68 and/or 217–232 were substituted with a GSN linker. In b, the Puf3-IDR was truncated and the indicated sequences were fused to the PUM RBD. c, Pulldown assays of truncated proteins used in b and Fig. 1b. MBP-tagged proteins were immobilized on amylose resin and tested for their ability to pull down Ccr4–Not. Fractional binding was quantified relative to the full-length protein. Bars denote the mean of three triplicate experiments (filled circles). d, Fluorescence anisotropy equilibrium analysis of Puf3 binding to labeled Ccr4–Not. Increasing concentrations of the indicated Puf3 constructs (109–258 and 158–258 are GST–lipoyl tag fusions with no PUM domain) were titrated into 25 nM S. pombe Ccr4–Not containing an FITC label on the Not9 subunit. Binding isotherms were fitted with a quadratic binding equation. Symbols represent the mean of three replicates with error bars showing the s.d. Calculated apparent KD values (±s.d.) are shown.
