Arginines 29 and 59 of elongation factor G are important for GTP hydrolysis or translocation on the ribosome

Abstract

GTP hydrolysis by elongation factor G (EF-G) is essential for the translocation step in protein elongation. The low intrinsic GTPase activity of EF-G is strongly stimulated by the ribosome. Here we show that a conserved arginine, R29, of Escherichia coli EF-G is crucial for GTP hydrolysis on the ribosome, but not for GTP binding or ribosome interaction, suggesting that it may be directly involved in catalysis. Another conserved arginine, R59, which is homologous to the catalytic arginine of G(alpha) proteins, is not essential for GTP hydrolysis, but influences ribosome binding and translocation. These results indicate that EF-G is similar to other GTPases in that an arginine residue is required for GTP hydrolysis, although the structural changes leading to GTPase activation are different.

Fig. 1. Effect of R29 and R59 mutations on turnover GTP hydrolysis by EF-G on the ribosome. (A) R29 mutations; (B) R59 mutations; (C) Michaelis–Menten titrations with R59 mutants. Wild-type EF-G (squares), R29K (open upward triangles), R29M (open downward triangles), R29A (open diamonds), R59K (filled upward triangles), R59M (filled downward triangles), R59A (filled diamonds).

Fig. 2. Kinetics of P_i release from EF-G following GTP hydrolysis on the ribosome. Upper panel, short time window; lower panel, long time window. EF-G or EF-G mutants (2 mM, final concentration) were rapidly mixed with ribosomes (0.2 mM) and liberated P_i was monitored by the fluorescence of MDCC-labeled PBP (Materials and methods). 1, wild-type EF-G; 2, R59K; 3, R59M; 4, R29A.

Fig. 3. Interaction of mant-GTP with R29 mutants of EF-G. The fluorescence change of mant-GTP upon binding to the EF-G mutants was measured by stopped flow. R29K (upward triangles), R29M (downward triangles), R29A (diamonds).

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).

Fig. 5. Translocation activity of EF-G R59 mutants. (A) Multiple-turnover translocation. EF-G wt (squares), R59K (upward triangles), R59M (downward triangles), R59A (diamonds), no EF-G (circles). (B) Single-round translocation. Time courses were evaluated by single-exponential fitting with the k_app values given. 1, wild-type EF-G(4.4 s^–1); 2, R59K (0.8 s^–1); 3, R59M (0.17 s^–1); 4, R59A (0.04 s^–1). (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). Experiments were performed as in Figure 4.

Fig. 6. Location of R31 (R29 in E.coli EF-G) in the crystal structure of EF-G from T.thermophilus. 1–5, domains of EF-G. A–E, helices in domain 1 (G domain). The positions of R31 and GDP are shown in space-filling representation. The location of R61 (R59 in E.coli EF-G) is not known, because this part of EF-G (effector region) is not resolved in the available crystal structures.