FIGURE 3.
Competition binding assays. Radiolabeled (A) unmodified tRNAPhe, (B) (2N3A76)tRNAPhe, (C) (2N32′OMeA76)tRNAPhe, (D) (2N3dA76)tRNAPhe, and (E) (2N3dA76)tRNAPhe-p-Puro (∼100 nM) were first mixed with various concentrations of unlabeled, unmodified tRNAPhe and then mixed with 70S ribosomes (10 nM) in the presence of polyU and 20 mM MgCl2. The binding reactions were incubated at 4°C for 48 h to reach equilibrium and then analyzed by the nitrocellulose filter binding assay. Error bars represent the standard deviation of triplicate samples of each reaction. The concentrations of competing unlabeled, unmodified tRNAPhe that displace 50% of the specifically bound radiolabeled ligand (IC50) were calculated using the nonlinear regression function of GraphPad Prism software. In these assays, radiolabeled ligands with higher affinities for the ribosome lead to higher IC50 values.