Complete kinetic mechanism for recycling of the bacterial ribosome

Abstract

How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec(-1), is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of ∼5 sec(-1), are discussed in the perspective of rapidly growing bacterial cells.

Keywords: bacterial ribosome recycling; elongation factor G; protein synthesis; ribosome recycling factor; translation rate optimization.

FIGURE 1.

Ribosome recycling time traces obtained at different EF-G and RRF concentrations and concentration dependencies of the recycling time. (A) Time traces for ribosome splitting obtained in the stopped-flow instrument at 5.1 µM EF-G and varying concentrations of RRF (1–40 µM). The fraction of post-termination complexes remaining in 70S form is plotted as a function of time. (B) Time traces for ribosome splitting obtained at 1 µM RRF and varying concentrations of EF-G, displaying the fraction of post-termination complexes remaining in 70S form as a function of time. (C) Recycling times obtained at 1.0, 5.1, or 10.2 µM EF-G plotted as a function of the RRF concentration. The fitted lines were calculated from Equation 2, with A-parameters determined from the full data set in Table 1 (see Equation 3) at the respective EF-G concentrations. The inset shows a Lineweaver–Burk plot (the inverse of the splitting rate, 1/k_rec = τ_rec, plotted versus the inverse of the RRF concentration, 1/[RRF]) of the same data set. (D) Recycling times obtained at 1, 3, or 20 µM RRF plotted as a function of the EF-G concentration for each RRF concentration. The fitted lines were calculated from Equation 2 at the respective RRF concentrations, as in C.

FIGURE 2.

The complete kinetic scheme for ribosome recycling. The definitions of the rate constants and their values are given in Table 2. RRF binds to the post-termination ribosomal complex with rate constant k_RRF and is released with rate constant q_RRF. EF-G binds to the RRF-free (X = 1) or the RRF-bound (X = 2) post-termination complex with rate constant k_GX. EF-G(GTP) can then either be released with rate constant q_G(GTP)X or hydrolyze GTP with rate constant k_GTPX. After GTP hydrolysis, EF-G(GDP) is released with rate constant q_GX. Once EF-G and RRF have bound to the ribosome it splits with rate constant k_split.

FIGURE 3.

GTP hydrolysis by EF-G on the post-termination complex in the absence of RRF. (A) The number of GTP molecules hydrolyzed by EF-G per active post-termination complex (0.17 µM) at various EF-G concentrations (0–40.8 µM) plotted as a function of time. [³H]-GTP was present at 1 mM, giving a maximal number of GTP hydrolysis cycles of about 5900. Straight lines were fitted to the data points at each EF-G concentration and the rate of hydrolysis per active post-termination complex was obtained from the slopes. (B) The rate of GTP hydrolysis obtained in A was plotted as a function of the EF-G concentration and fitted to the Michaelis–Menten equation. The (k_cat)_G1 value was determined as 36 ± 0.3 sec⁻¹ and (K_M)_G1 value as 0.56 ± 0.02 µM⁻¹ sec⁻¹.

FIGURE 4.

Determination of the number of GTP molecules consumed per ribosome splitting event. (A) Time curves of ribosome splitting obtained in the presence of 1 µM post-termination complexes, 0.5 µM EF-G, 20 µM [³H]-GTP, and different concentrations of RRF. At each RRF concentration, the concentration of split ribosomes, calculated from the fluorescence intensity as described in Materials and Methods, was plotted as a function of time. The points indicate the concentration of split ribosomes at different incubation times, calculated from fits of two-exponential functions to the splitting curves. The curve obtained at 2 µM RRF had lower amplitude because GTP was running out before complete splitting was achieved. (B) Time curves of GTP hydrolysis obtained under the same conditions as in A. At each RRF concentration, the concentration of hydrolyzed GTP was plotted as a function of time. The fitted lines are single exponentials followed by a straight line and are just indicative, as it was the extent of GTP hydrolysis measured at different times of incubation, indicated by the points, that was used for further analysis. (C) The ratio of the concentration of GTP hydrolized in B and the concentration of split ribosomes in A plotted as a function of time at the respective RRF concentrations. Straight lines were fitted to the data points of which the intercepts reflect the number of GTP molecules consumed per splitting event. The slopes reflect GTP hydrolysis by EF-G on 50S subunits formed during the reaction. (D) The number of GTP molecules per splitting event plotted as a function of the RRF concentration and fitted to Equation 18. The inset shows the same data, the number of GTPs consumed per splitting event, as a function of the inverse of the RRF concentration. The intercept of the straight line fitted to the data reflects the minimal number of GTP molecules consumed per splitting event at very high RRF concentrations and is 1.1 ± 0.1.

FIGURE 5.

The ribosome recycling rate and the number of GTP molecules consumed per splitting event as a function of the EF-G and RRF concentrations. (A) Simulation of the recycling rate according to Equation 1 at 0–21 µM EF-G and 0–40 µM RRF. The rate of recycling, plotted as a function of the RRF and EF-G concentrations, increases in a Michaelis–Menten-dependent manner with increasing RRF concentration at all EF-G concentrations. Note that in this concentration range rates are far below their maximum (25 sec⁻¹). With respect to EF-G, there is an optimal concentration at each RRF concentration yielding a maximal recycling rate. (B) Simulation of the number of GTP molecules consumed per splitting event according to Equation 18 at 0–21 µM EF-G and 0–40 µM RRF. The number of GTPs consumed per ribosome splitting event is plotted as a function of the RRF and EF-G concentrations. At constant RRF concentration the GTP consumption increases linearly with the EF-G concentration, whereas at constant EF-G concentration the GTP consumption decreases hyperbolically with the RRF concentration.