32P-labeled 5′UTRPab1 and proteins bound—including eIF4G1, Pab1, Sbp1, and its domain variants—were mixed at different stoichiometric ratios. Binding of the RNA to the protein was examined by electrophoresis in an 8% (bis-acrylamide 29:1) polyacrylamide under native conditions in THEM buffer (34 mM Tris Base, 57 mM Hepes, 0.1 mM EDTA, 2.5 mM MgCl2). Bound and unbound RNAs were quantified using ImageQuant software (Molecular Dynamics). The dissociation constant (Kd) and the Hill coefficient (n) was obtained by fitting data to the following equation: [RNA]bound([RNA]bound+[RNA]free)=[P]totaln(Kdn+[P]totaln), where [P]total is the total concentration of the protein, [P]total ≈ [P]free under the conditions used, and [RNA]bound and [RNA]free are concentrations of RNA in the protein-bound and free forms, respectively.