Control of phosphate release from elongation factor G by ribosomal protein L7/12 - PubMed (original) (raw)

Control of phosphate release from elongation factor G by ribosomal protein L7/12

Andreas Savelsbergh et al. EMBO J. 2005.

Abstract

Ribosomal protein L7/12 is crucial for the function of elongation factor G (EF-G) on the ribosome. Here, we report the localization of a site in the C-terminal domain (CTD) of L7/12 that is critical for the interaction with EF-G. Single conserved surface amino acids were replaced in the CTD of L7/12. Whereas mutations in helices 5 and 6 had no effect, replacements of V66, I69, K70, and R73 in helix 4 increased the Michaelis constant (KM) of EF-G.GTP for the ribosome, suggesting an involvement of these residues in EF-G binding. The mutations did not appreciably affect rapid single-round GTP hydrolysis and had no effect on tRNA translocation on the ribosome. In contrast, the release of inorganic phosphate (Pi) from ribosome-bound EF-G.GDP.Pi was strongly inhibited and became rate-limiting for the turnover of EF-G. The control of Pi release by interactions between EF-G and L7/12 appears to be important for maintaining the conformational coupling between EF-G and the ribosome for translocation and for timing the dissociation of the factor from the ribosome.

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Figures

Figure 1

Positions of mutations in the CTD of protein L7/12 from E. coli. Amino acids are numbered according to the sequence of the protein as in the crystal structure (Leijonmarck and Liljas, 1987).

Figure 2

Effect of L7/12 mutations on turnover reactions of EF-G. (A) GTP hydrolysis. Time courses were measured with native ribosomes (•), ribosomes reconstituted with wt L7/12 (○), R73K (⧫), V66A (□), R73 M (⋄), I69A (▾), K70A (▴), or V66D (▪), and ribosome cores lacking L7/12 (*). EF-G (0.04 μM) was incubated with [γ-32P]GTP (20 μM) and ribosomes (0.2 μM) at 37°C. (B) Translocation. Pretranslocation complex carrying fMetPhe-tRNAPhe in the A site (0.2 μM) was incubated with EF-G (0.5 nM) and GTP (1 mM) at 37°C. At the indicated times, puromycin (Pmn; 1 mM) was added and the reaction stopped after 10 s. The extent of fMetPhe-Pmn formation is given relative to the initial amount of pretranslocation complex. Symbols as in panel A.

Figure 3

Effect of L7/12 mutations on single-round GTP hydrolysis and translocation catalyzed by EF-G. (A) Time courses of GTP hydrolysis. EF-G and [γ-32P]GTP were rapidly mixed with ribosomes (Materials and methods). Unlabeled GTP (1 mM) was added together with the ribosomes in order to limit [γ-32P]GTP hydrolysis to a single round (Rodnina et al, 1997). Symbols as in Figure 2. (B) Rate constants of GTP hydrolysis. Time courses shown in panel A were evaluated by exponential fitting to yield rate constants as indicated. (C) Time courses of translocation as monitored by the fluorescence of fMetPhe-tRNAPhe(Prf16/17). Overlaid stopped-flow traces are shown for ribosomes reconstituted with wt L7/12, V66A, V66D, I69A, K70A, R73M, and R73K.

Figure 4

Pi release from EF-G after GTP hydrolysis. (A) Time courses of Pi release. EF-G (3 μM) and GTP (30 μM) were mixed with ribosomes (0.5 μM) in the stopped-flow apparatus and the liberation of Pi was monitored by the fluorescence change of MDCC-labeled PBP (Materials and methods). Traces are shown for ribosomes reconstituted with wt L7/12 (1), R73K (2), R73M (3), V66A (4), I69A (5), K70A (6), V66D (7) and for ribosome cores depleted of L7/12 (8). Exponential fitting yielded burst rates of 20±10 s−1 in all cases, except for cores depleted of L7/12 where the burst phase is lacking. The rates of the slower turnover phase were 3–4 s−1 (wt, R73M, R73K), 2 s−1 (V66A, I69A), or 1 s−1 (V66D, K70A). The final level of all time courses was identical within 10% and was normalized to 1.0. (B) Relative burst amplitudes of single-round GTP hydrolysis (closed bars) and Pi release (open bars). The extent of [γ-32P]GTP hydrolysis was determined either from quench-flow data (Figure 3) or from the amount of [γ-32P]GTP hydrolyzed after 10 s at conditions where multiple turnover was suppressed by the addition of unlabeled GTP. Amplitudes of Pi release were estimated from the data in panel A. Burst amplitudes for GTP hydrolysis and Pi release determined for ribosomes reconstituted with wt L7/12 were set to 1.0 and relative numbers are given for cores and reconstituted mutant ribosomes. (C) Kinetic scheme of translocation used for the evaluation of rate constants of Pi release (Savelsbergh et al, 2003). Binding of EF-G to the ribosome is followed by rapid GTP hydrolysis (50–200 s−1, cf. Figure 3B); a conformational change (‘unlocking'; 10–35 s−1; Savelsbergh et al, 2003) precedes and limits (fully or partially) the following tRNA–mRNA translocation. Translocation (or a structural rearrangement accompanying tRNA movement) and Pi release are parallel and independent of one another. The rate of Pi release is decreased to 1–2 s−1 by mutations at positions 66, 69, and 70 of the L7/12 CTD. Subsequent conformational changes of the ribosome and EF-G are important for EF-G dissociation from the ribosome.

Figure 5

Time courses of Pi release from EF-Tu. Purified ternary complex EF-Tu·GTP·Phe-tRNAPhe (1 μM) was rapidly mixed with poly(U)-programmed ribosomes (1 μM) containing AcPhe-tRNAPhe in the P site. Traces are shown for native ribosomes (1) and ribosomes reconstituted with wt L7/12 (2), R73M (3), K70A (4), or K84A (5).

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Control of phosphate release from elongation factor G by ribosomal protein L7/12 - PubMed (original) (raw)