Fig. 4. Translocation activity of R29 mutants of EF-G. (A) Multiple-turnover translocation. Pretranslocation complex (0.2 µM) was incubated with wild-type EF-G (squares), R29K (upward triangles), R29M (downward triangles), R29A (diamonds) (0.04 µM) or without factor (circles) in the presence of GTP (1 mM). Translocation was measured by the formation of fMetPhe-puromycin. (B) Single-round translocation measured by stopped flow. Pretranslocation complex containing fMetPhe-tRNA^Phe(Prf16/17) in the A site (0.1 µM, final concentration) was rapidly mixed with EF-G (0.8 µM) in the presence of GTP (1 mM), caged GTP (0.1 mM) or without nucleotide. Time courses were evaluated by single-exponential fitting with the k_app values given. 1, wild-type EF-G (4.4 s^–1); 2, wild-type EF-G with caged GTP (0.5 s^–1); 3, R29M (0.4 s^–1); 4, R29K (0.15 s^–1); 5, R29A (0.11 s^–1); 6, R29K without nucleotide; EF-G(R29M) and EF-G(R29A) without nucleotide were the same (not shown). (C) Dependence of k_app of translocation on the concentration of mutant and wild-type (inset) EF-G. For k_app values measured at 2 µM factor see Table II. Symbols are as in (A).