Stepwise translocation experiments were done as previously described (15) . Tight-couple 70 S ribosomes (1 mM final concentration) from E. coli MRE600 (39) were incubated with mSP-HIV-1 mRNA (1 mM) in 30 ml binding buffer (10 mM Tris-HCl (pH 7.4), 60 mM NH 4 Cl, 10 mM Mg(OAc) 2 , 6 mM b-ME) for 10 min at 37 C, followed by addition of tRNA Phe (1 mM) and a further 10 min incubation to fill the P site. Aliquots (5 ml) of this reaction were then added to separate tubes containing either GTP (600 mM) (F), GTP + tRNA Leu (1 mM) (L), GTP + tRNA Leu , EF-G (1 mM) (L'), GTP + tRNA Leu , EF-G + tRNA Gly (1 mM) (G), GTP + tRNA Leu , EF-G + tRNA Gly + tRNA Lys (1 mM) (K), GTP + tRNA Leu + tRNA Gly + tRNA Lys (K'), in binding buffer (final volume, 10 ml). These tubes were incubated for 10 min at 37 C and then placed on ice for the primer extension in toeprinting analysis (4 ml).