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. 2009 Jul 22;28(14):2053-65.
doi: 10.1038/emboj.2009.169. Epub 2009 Jun 18.

Conformational changes in switch I of EF-G drive its directional cycling on and off the ribosome

Cristina Ticu  1 , Roxana Nechifor, Boray Nguyen, Melanie Desrosiers, Kevin S Wilson
Affiliations

Conformational changes in switch I of EF-G drive its directional cycling on and off the ribosome

Cristina Ticu et al. EMBO J. .

Abstract

We have trapped elongation factor G (EF-G) from Escherichia coli in six, functionally defined states, representing intermediates in its unidirectional catalytic cycle, which couples GTP hydrolysis to tRNA-mRNA translocation in the ribosome. By probing EF-G with trypsin in each state, we identified a substantial conformational change involving its conserved switch I (sw1) element, which contacts the GTP substrate. By attaching FeBABE (a hydroxyl radical generating probe) to sw1, we could monitor sw1 movement (by approximately 20 A), relative to the 70S ribosome, during the EF-G cycle. In free EF-G, sw1 is disordered, particularly in GDP-bound and nucleotide-free states. On EF-G*GTP binding to the ribosome, sw1 becomes structured and tucked inside the ribosome, thereby locking GTP onto EF-G. After hydrolysis and translocation, sw1 flips out from the ribosome, greatly accelerating release of GDP and EF-G from the ribosome. Collectively, our results support a central role of sw1 in driving the EF-G cycle during protein synthesis.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Six states of EF-G, free or ribosome-bound. (A) The EF-G catalytic cycle. Boxes represent tRNA interaction sites (A, P, and E) on the 30S and 50S subunits of the ribosome. The EF-G interaction sites are designated by F. Rotation of the ribosomal subunits is represented by the shift in boxes. The six trapped states are highlighted by the red EF-G cartoons and are identified by bold lettering. (B, C) GTP hydrolysis assays. Panel B: single-turnover conditions: EF-G (0.8 μM), GTP (0.5 μM, with trace radioactive [γ32-P]GTP), and vacant ribosome (1.0 μM). Panel C: multiple-turnover conditions: EF-G (0.04 μM), GTP (50 μM), and vacant ribosome (0.5 μM). Inhibitors: GDPNP (100 μM), vio (250 μM), or fus (200 μM). Reactions (37°C, 20 min) were quenched with formic acid (30%) and resolved by thin layer chromatography (Nechifor and Wilson, 2007). (D) Extent of ribosome translocation, monitored by the toeprinting assay (Wilson and Nechifor, 2004). (E) Kinetics of ribosome translocation, measured by using 3′-pyrene-mRNA (Studer et al, 2003; Nechifor et al, 2007). Fluorescence traces (offset for clarity) monitor translocation kinetics. In Panels D and E, reactions contained: pretranslocational ribosome (0.5 μM; see Materials and methods), EF-G (1.0 μM), and GTP (1 mM). Inhibitors were GDPNP (1 mM), vio (0.5 mM), or fus (1 mM).
Figure 2
Figure 2
Trypsin preferentially cleaves sw1 in EF-G•GDP, free or ribosome-bound. (A) Free EF-G states, probed with trypsin. Complexes were formed with EF-G (2 μM) containing GDP (0.5 mM), GTP (0.5 mM), or no nucleotide. Trypsin (4.5 μg/ml) was added, and reactions were incubated (20°C). Samples of each reaction were removed and denatured (after 1, 4, 16, and 64 min), and analysed by SDS–PAGE (Materials and methods). (B) Stability of ribosome-bound EF-G complexes. Step (1): EF-G (1.5 μM) was bound to a pretranslocational ribosome (1.5 μM) in the GDPNP, GDP/fus, or GDP/vio states (Materials and methods). Step (2): free EF-G was removed from ribosome-bound EF-G by filtration (Materials and methods). Step (3): complexes were treated with trypsin (4.5 μg/ml; 20°C; 10 min). Step (4): reactions were quenched with trypsin inhibitor (340 μg/ml), and the digested complexes were re-purified by filtration. (C) Ribosome-bound EF-G states, probed with trypsin. Complexes were formed with EF-G (1.7 μM) bound to a pretranslocational ribosome (2.0 μM) in GDPNP, GDP/vio, and GDP/fus states (Figure 1). After removing free EF-G, complexes were probed with trypsin and analysed by SDS–PAGE as in panel A. (D) Peptide bond cleaved by trypsin in the sequence of sw1 of E. coli EF-G, deduced from the N-terminal sequence of the 72-kDa fragment (Supplementary Table 1).
Figure 3
Figure 3
Sw1 moves relative to ribosome-bound EF-G, after GTP hydrolysis. (A) Formation of ribosome complexes. N-acetyl-Phe-tRNAPhe (2.4 μM) was bound to the P site of a vacant (v) ribosome (2.0 μM), containing T4 gene 32 mRNA. The resulting complex was split into two equal volumes: one was left untreated; the other was treated with puromycin (1 mM; 20°C, 30 min; 37°C, 8 m), forming N-acetyl-Phe-puromycin (released from the ribosome) and deacylated tRNAPhe (remaining on the ribosome). Before treatment, the ribosomal subunits are expected to adopt their unrotated (u) conformation (Valle et al, 2003), and the tRNA binds in the P/P state (Moazed and Noller, 1989). After treatment, the subunits can convert to their rotated (r) conformation, and the deacylated tRNAPhe can move to its P/E state. (B) Chemical probing of tRNA movement from P/P to P/E states in the ribosome. The three ribosomes (v, u, and r; panel A) were probed with kethoxal, which chemically modifies G2252 and G2253 of 23S rRNA in the 50S P site. Their modification was monitored by primer extension (Stern et al, 1988). tRNA occupation of the P/P state and movement to the P/E state are indicated by the relative protection and exposure (respectively) of G2252 and G2253 (Samaha et al, 1995). (C) Chemical probing of EF-G binding to the ribosome. EF-G (1.7 μM) was bound to complexes u and r in the GDP/fus state. The resulting complexes were purified from free EF-G by filtration. EF-G interaction with the 50S F site was probed with dimethyl sulfate, which chemically modifies nucleotide A2660 in 23S rRNA (Moazed et al, 1988). EF-G binding is indicated by the protection of A2660. (D) Enzymatic probing of EF-G. EF-G-containing ribosome complexes were exposed to trypsin, and analysed by SDS–PAGE (as in Figure 2C).
Figure 4
Figure 4
Sw1 flips out from the ribosome cavity, after GTP hydrolysis and 30S translocatiuon. (A) Tracking sw1 movement with FeBABE. Three derivatives of E. coli EF-G (single Cys mutants 58C and 196C, and Cys-free) were treated with FeBABE (Supplementary data). Proteins 58C-FeBABE and 196C-FeBABE (2 μM) were bound to a pretranslocational ribosome (0.5 μM) in GDPNP, GDP/fus, and GDP/vio states (Figure 1; Materials and methods). The control FeBABE-treated Cys-free protein was bound in the GDP/fus state. All seven complexes were treated with hydrogen peroxide and ascorbate (Materials and methods) to generate localized hydroxyl radicals from the iron atom of the FeBABE probe (Wilson and Noller, 1998). After quenching radicals with thiourea, rRNAs were extracted from the complexes. Strand cleavages in 16S and 23S rRNA were analysed by primer extension (Stern et al, 1988; Wilson and Nechifor, 2004). Shown here is a primer extension analysis of the region including helix 95 of 23S rRNA. Lanes A, G: sequencing reactions. Numbers (left) refer to E. coli 23S rRNA nucleotides. Rectangles (right) identify strongly cleaved rRNA nucleotides, and are colour-coded as follows: magenta: 58C-FeBABE in GDPNP and GDP/vio states; blue: 58C-FeBABE in GDP/fus state. (B) rRNA nucleotides targeted by 58C-FeBABE. Magenta and blue spheres are centred on Pα atoms of cleaved rRNA nucleotides (panel A; Supplementary Figure 5), and are colour-coded as in panel A. Red and black spheres are centred on Cα atoms of EF-G residues 58 and 196, respectively. Sw1 and G domain are coloured red and yellow, respectively. The overall structural framework is based on T. thermophilus ribosome-bound EF-G•GDPNP (Connell et al, 2007). Superpositioned onto it are the structures of EF-G-2•GDPNP (Connell et al, 2007) and EF-G(G16V)•GDP (Hansson et al, 2005b). Arrow shows the movement of residue 58 between the superpositioned structures. (C) Sw1 conformation in the GDPNP state. The view is orthogonal to that of panel B. The ribosome is invisible except for nucleotides in helix 95 cleaved by 58C-FeBABE. (D) Sw1 conformation in the GDP/fus state.
Figure 5
Figure 5
Kinetics of release of nucleotide and EF-G from the ribosome. (A) Release of fluorescent nucleotide from ribosome-bound EF-G. Two complexes were formed with wild-type E. coli EF-G (2 μM), vacant ribosome (3 μM), and either mant-GDPNP (2 μM; blue dots) or mant-GDP (2 μM; red dots). These complexes were rapidly mixed with excess, unlabelled GDPNP or GDP (2.4 mM). See ‘Materials and methods'. The inserted graph focuses on the first second of the two reactions after mixing, in order to show more clearly the rapid release of mant-GDP. (B) Release of fluorescent EF-G from the ribosome. Two complexes were formed with EF-G(OG) (2 μM), vacant ribosome (3 μM), and either GDPNP (200 μM; blue dots) or GDP (200 μM; red dots). These complexes were rapidly mixed with excess, wild-type E. coli EF-G (20 μM). See ‘Materials and methods'. Likewise to panel A, the inserted graph focuses on the first 0.4 sec of the reactions.
Figure 6
Figure 6
Model of sw1 conformational changes, driving the EF-G catalytic cycle. (A) Sw1 in ribosome-bound EF-G locks GTP in the ribosome. Structure is based on T. thermophilis EF-G•GDPNP•ribosome (Connell et al, 2007). Its electron density (mesh, contoured at 3σ) and underlying molecular model (ribbon) are shown in white. Highlighted elements in this model include sw1 (red), GDPNP (cyan), G domain of EF-G (yellow), and helix 95 of 23S rRNA (pink). (B) Flipped-out sw1 permits the escape of GDP and Pi after GTP hydrolysis. Represented here is ribosome-bound EF-G, before and after GTP hydrolysis. The view is orthogonal to that in panel A, and only domain I of EF-G is shown for clarity. Two structures are superpositioned onto ribosome-bound EF-G: Left structure (before GTP hydrolysis) is T. thermophilus EF-G-2-GDPNP (Connell et al, 2007); right structure (after hydrolysis) is E. coli EF-G(G16V)•GDP (Hansson et al, 2005b). Colour scheme is described in panel A, except that GDP here is dark blue and Pi is cyan. (C) Proposed mechanism for sw1 conformational changes occurring within the EF-G cycle. The general format here follows Figure 1A. Proceeding left to right, the diagram starts with EF-G bound to the ribosome, after subunit rotation and before GTP hydrolysis. The squiggly line represents the nascent polypeptide chain attached to tRNA in the A/P hybrid state. Sw1 is represented by the red flap. Sw1 flips out from the ribosome after GTP hydrolysis (centre diagram). The diagram ends with release of EF-G from the ribosome, and the cycle is completed with a fresh GTP substrate binding to EF-G off the ribosome.
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