Contamination of GDP preparations with GTP strongly stimulates translocation by EF-G. (a) Elution profile of commercially available GDP from a MonoQ column showing the GTP and GMP contaminations. %B is the percentage of buffer B (20 mM Tris-HCl, 1 M NaCl) in the buffer A (20 mM Tris-HCl) + B mixture. (b) Time-dependent release of peptide by 0.4 μM RF2 after translocation of fMet-Ile-tRNA (23 nM total) from the A site to the P site by 1 μM EF-G in the presence of 1 mM purified GDP, unpurified GDP, purified GDP containing 20 μM GTP (2%), or 20 μM GTP. (c) Cleavage of mRNA by RelE incubated with 0.15 μM preT, 2 μM EF-G and nucleotides. Lanes: (1) no GDP; (2) 1 mM purified GDP; (3) 1 mM unpurified GDP; (4) 1 mM purified GDP containing 2% GTP; (5) 20 μM GTP.