Elongation factor G initiates translocation through a power stroke

Abstract

During the translocation step of prokaryotic protein synthesis, elongation factor G (EF-G), a guanosine triphosphatase (GTPase), binds to the ribosomal PRE-translocation (PRE) complex and facilitates movement of transfer RNAs (tRNAs) and messenger RNA (mRNA) by one codon. Energy liberated by EF-G’s GTPase activity is necessary for EF-G to catalyze rapid and precise translocation. Whether this energy is used mainly to drive movements of the tRNAs and mRNA or to foster EF-G dissociation from the ribosome after translocation has been a long-lasting debate. Free EF-G, not bound to the ribosome, adopts quite different structures in its GTP and GDP forms. Structures of EF-G on the ribosome have been visualized at various intermediate steps along the translocation pathway, using antibiotics and nonhydolyzable GTP analogs to block translocation and to prolong the dwell time of EF-G on the ribosome. However, the structural dynamics of EF-G bound to the ribosome have not yet been described during normal, uninhibited translocation. Here, we report the rotational motions of EF-G domains during normal translocation detected by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy. Our study shows that EF-G has a small (∼10°) global rotational motion relative to the ribosome after GTP hydrolysis that exerts a force to unlock the ribosome. This is followed by a larger rotation within domain III of EF-G before its dissociation from the ribosome.

Dynamic relationships between ribosomal conformational and RNA positional changes during ribosomal translocation

“…In other words, the binding of EF-G hydrolyzing GTP is only purposed to facilitate the ribosomal unlocking, allowing the movement of mRNA-tRNA complex in the 30S subunit, and it does not provide the forward force to facilitate tRNA movement or act as a pawl to block the reverse movement of tRNA after the translocation. These are consistent with the recent single molecule data on conformational changes of EF-G in the ribosome [23], as discussed as follows.…”

“…The flexible rotations of domain III to the large extent, which occur after the A-site tRNA movement to the P site, are an intrinsic property of EF-G in GDP form [38], which can be noted from the single molecule evidence that when EF-G.GTP binds to the ribosomal complex in the absence of A-site tRNA, where no translocation can occur [39] (see, Refs. [40] for detailed discussion), the distributions of rotation angles for EF-G domains were quite similar to those when EF-G-GTP binds to pretranslocation complex with two tRNAs [23]. This can also be noted from the single molecule evidence that the antibiotics viomycin (Vio) and spectinomycin (Spc) considerably reduced the extent of rotations in EF-G domain III but had little or even no effect on rotations of domains I, IV and V in pretranslocation complex with two tRNAs [23], because the A-site tRNA restricts flexible rotations of domain III to the large extent.…”

“…This can also be noted from the single molecule evidence that the antibiotics viomycin (Vio) and spectinomycin (Spc) considerably reduced the extent of rotations in EF-G domain III but had little or even no effect on rotations of domains I, IV and V in pretranslocation complex with two tRNAs [23], because the A-site tRNA restricts flexible rotations of domain III to the large extent. By contrast, when the A site was empty, neither Vio nor Spc had perceptible effects on the motion of EF-G domain III [23], because no A-site tRNA restricts the motion although no translocation occurs.…”

“…The single molecule data showed that EF-G.GTP binding to the pretranslocation complex bound with two tRNAs is followed by a small (∼10°) global rotational motion of EF-G domains I, IV and V relative to the ribosome and by contrast, domain III consistently showed very large and variable rotational motions, whose angular changes were evenly distributed from 0° to 90° without a clear peak [23]. The small global rotational motion of EF-G domains I, IV and V facilitates the ribosomal unlocking via 30S head rotation.…”

“…This results in State H transiting to another hybrid state (State H1), which causes the peptidyl-tRNA to change the position in 50S subunit, with FRET between Cy3-labeled ribosomal protein L11 and Cy5-labeled peptidyl-tRNA (L11-t FRET) decreasing from 0.6 to 0.4, with the rate of reactivity of peptidyl-tRNA toward puromycin (denoted by Pmn) increasing from the order of ${10}^{-3}$ ${\text{s}}^{-1}$ to the order of 0.1–1 ${\text{s}}^{-1}$ and with the peptidyl-tRNA becoming closer to deacylated tRNA. In State H1 with EF-G in GDP.Pi form, small conformational change in EF-G or small global rotational motion of EF-G relative to the ribosome causes the tip (loops I and II) of domain IV to move towards and interact with the decoding center in the 30S subunit, facilitating the forward rotation of the 30S head relative to the 30S body (State H2) [7, 23]. The forward 30S head rotation then leads to widening of the mRNA channel or tilting of the 30S head (State H3) [7, 8, 9, 10, 11, 12, 24], which is termed as ribosomal unlocking.…”