Aminoacyl-tRNA synthetases were purified as described earlier (22, 23) .Yeast tRNA Phe was aminoacylated with unlabeled phenylalanine or with [ 14 C]Phe to make Phe-tRNA Phe and [ 14 C]Phe-tRNA Phe , respectively. [ 14 C]Phe-tRNA Phe was used to monitor enzymatic binding to the A site of poly(U) primed ribosomes, and acetylated-[ 14 C]Phe-tRNA Phe (Ac-[ 14 C]Phe-tRNA Phe ) was generated to monitor nonenzymatic P-site binding using poly(U) primed salt washed ribosomes. Phe-tRNA Phe and Ac-Phe-tRNA Phe were used in SHAPE structure probing experiments (see below). Yeast tRNA Phe was aminoacylated as described earlier (24) Reaction mixtures were incubated for 30 min at 30 C, and proteins were removed by extraction with acid-phenol-chloroform. Charged tRNAs were ethanol precipitated and purified using G25 spin columns. [ 14 C]Phe-tRNA Phe was separated from uncharged tRNA by high-performance liquid chromatography (HPLC) as described earlier (25) with the following modifications. Samples were loaded onto a 4.6-by 250-mm JT Baker wide-pore butyl column equilibrated with buffer A (20 mM NH 4 Cl, 10 mM MgCl 2 , 400 mM NaCl; pH 5.0) at 1 ml/min. The column was washed with 10 ml of buffer A, conditions under which free phenylalanine and aminoacyladenylate are eluted from the column. Uncharged tRNAs and residual free [ 14 C]Phe and nucleotides were eluted by isocratic elution of 19 ml at 15% of buffer B (20 mM NH 4 Cl, 10 mM MgCl 2 , 400 mM NaCl, 60% methanol; pH 5.0). [ 14 C]Phe-tRNA Phe was eluted using a programmed binary gradient of buffers A and B. Elution of aa-tRNA was monitored by OD 260 readings, and [ 14 C]Phe-tRNA Phe concentrations and specific activities were determined. The presence of aa-tRNA in the eluted material was confirmed by TLC (24) . Ac-[ 14 C]Phe-tRNA Phe was obtained in a similar manner. Yeast tRNA Phe was charged with [ 14 C]Phe as above, extracted with phenol and purified using G25 columns. Reaction mix (4 ml) contained 200 mM NaOAc, pH 5.2 and 7 nmol of [ 14 C]Phe-tRNA Phe . Acetylation was carried out by addition of 64 ml of acetic anhydride at 1 h intervals for 2 h on ice. After incubation, NaOAc concentration was raised to 300 mM and Ac-[ 14 C]Phe-tRNA Phe was ethanol precipitated. Ac-[ 14 C]Phe-tRNA Phe was further purified by HPLC as described earlier.