His-tagged or GST-tagged eIF1, eIF1A, Sbp1, and Pab1 and their mutants containing individual domains were overexpressed in E. coli BL21 (DE3) with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 18°C. The proteins were purified by affinity chromatography using either Ni-NTA or glutathione agarose, which is followed by ion-exchange chromatography and HiTrap Heparin HP columns (GE Healthcare). Circular dichroism (CD) was performed on the wild-type Sbp1 and its individual RRM domains to confirm proper folding of the RRM domain mutants. eIF2 was expressed in yeast and purified as previously described (Acker et al. 2007). A TAP tag with a calmodulin binding peptide and two IgG binding domains of protein A (Dharmacon) was inserted at the end of the eIF4G1 gene in its chromosome locus. The C-terminal TAP-tagged eIF4G1 was expressed in yeast cells and purified with affinity chromatography using buffer containing 20 mM Hepes-KOH, pH 7.9, 150 mM KCl, 1 mM Mg(OAc)2, 0.1% NP-40, 1 mM DTT followed by a HiTrap Heparin HP column (GE Healthcare) using buffer containing 20 mM Tris–HCl, pH 7.5, 150 mM KCl, 0.1% NP-40. RNase A and high salt wash with 1 M KCl were used to remove endogenous RNAs bound to eIF4G1.