The translocation of mRNA was assayed by toeprinting as described (35, 36) . mRNA (1 mM) was annealed to primer (2 mM) in 50 mM K-Hepes (pH 7.0) and 100 mM KCl by heating to 90 C for 1 min and placing at room temperature until the temperature reached 45 C. To form the complexes, tight-couple ribosomes (2-5 mM) (37) from E. coli MRE600 were added to 0.6 mM of mRNA in 60 mM NH 4 Cl, 10 mM Tris-Acetate (pH 7.4) and 20 mM MgCl 2 and incubated at 37 C for 10 min. A first tRNA (4 mM) was added to fill the P site by incubation at 37 C for 10 min and aliquot (0.6 pmol mRNA) was removed to ice for later extension. A second tRNA (4 mM) was added to fill the A site by incubation at 37 C for 10 min and aliquot (0.6 pmol mRNA) was removed to ice for later extension. EF-G was added in buffer (50 mM Tris-HCl (pH 7.6), 20 mM MgCl 2 , 100 mM NH 4 Cl and 1 mM DTT, 1.5 mM GTP) such that the final concentrations of GTP and EF-G were 300 and 1 mM, respectively. Reactions were incubated at 37 C for 10 min, and aliquots (0.6 pmol mRNA) were removed from each reaction lacking (-G) or containing (+G) EF-G. Each of the aliquots was then extended in parallel (38) with (5 0 -CTTTATCTTCAGAAGAAAAACC-3 0 ) primer, and the product were resolved by 8% denaturing PAGE.