Soluble protein factors were prepared as described earlier (28, 29) . aa-tRNA binding to the A-site of the ribosome was carried out as described earlier (13) . Ribosome mixtures (50 ml) contained 80 mM Tris-HCl, pH 7.4, 160 mM NH 4 Cl, 11 mM Mg(CH 3 COO) 2 ]Phe-tRNA Phe were added to ribosome mixtures and incubated for 20 min at 30 C. Aliquots were then applied onto nitrocellulose membranes, filters were washed with 6 ml of binding buffer, and radioactivity was measured by scintillation counting. Background levels of radioactivity were determined using a blank sample (without ribosomes) and subtracted from test samples. K d values were determined assuming single binding sites using Graphpad Prism software. eEF2 binding 6xHis-tagged eEF2 was purified from TKY675 yeast cells (kindly provided by Dr T. Kinzy) as described earlier (30) with the following modifications. EDTA was added to 5 mM to eluted eEF2 just before dialysis to bind leached Ni 2+ ions and prevent precipitate formation during dialysis due to aggregation of His-tagged protein. eEF2 concentration was determined by [ 14 C]ADP-ribosylation with diphtheria toxin (see below). Each preparation of eEF2 showed linear concentration response curves in the range of eEF2 amounts used in binding experiments. For eEF2-binding experiments, reaction mixes (25 ml) containing 12.5 pmol of salt washed 80S ribosomes and various concentrations of 6xHis-tagged eEF2 in binding buffer (50 mM Tris-HCl, pH 7.5, 50 mM ammonium acetate, 10 mM magnesium acetate, 2 mM DTT, 100 mM GDPNP) were incubated for 20 min at room temperature. Estimation of bound eEF2 was carried out as follows by assuming that ribosome bound eEF2 is not susceptible to ADP-ribosylation by diphtheria toxin (31) (32) (33) . Free (unbound) eEF2 was estimated by ADP-ribosylation of eEF2: 100 pmol [ 14 C] NAD + and 0.2 mg of diphtheria toxin were added to each reaction mix and incubated for 30 min at 30 C. Total eEF2 in each reaction mix was determined by ADP-ribosylation reaction after bound eEF2 was released by adding EDTA to 10 mM. Reaction mixes were precipitated with TCA, and amounts of [ 14 C]ADP-ribosylated eEF2 were determined by liquid scintillation counting. Control values (lacking diphtheria toxin) were subtracted. Ribosome bound eEF2 was calculated by subtracting free values from total amount. K d values were determined assuming single binding sites using Graphpad Prism software.