Cells were harvested at OD600 = 0.85–0.9 and washed five times with translation buffer comprised of 22 mM Hepes-KOH, pH 7.4, 120 mM potassium acetate, 1.5 mM magnesium acetate, 0.75 mM ATP, 0.1 mM GTP, 25 mM creatine phosphate (Invitrogen), 0.04 mM of each of the twenty amino acids, 1.7 mM 1,4-dithiothreitol (DTT), 5 µg creatine kinase (Invitrogen), and 10 U RNasin Plus (Promega). Cell extracts were treated with micrococcal nuclease and prepared as previously described (Iizuka et al. 1994). An m7G cap was added at the 5′ end of the RNA using the Vaccinia capping enzyme (NEB). In vitro translation reactions were carried out at 25°C for 40 min in translation buffer. m7G-capped or uncapped RNAs were added to the extracts in the presence of Sbp1 and its domain variants at varying concentrations, and luciferase activities were measured using the ONE-Glo Luciferase Assay kit (Promega). The measured reporter activity was normalized to the concentration of total proteins in the extract obtained by Bradford assay. All reported activities are an average of at least three independent luciferase assays.