Cryo-EM shows stages of initial codon selection on the ribosome by aa-tRNA in ternary complex with GTP and the GTPase-deficient EF-TuH84A

Abstract

The GTPase EF-Tu in ternary complex with GTP and aminoacyl-tRNA (aa-tRNA) promotes rapid and accurate delivery of cognate aa-tRNAs to the ribosomal A site. Here we used cryo-EM to study the molecular origins of the accuracy of ribosome-aided recognition of a cognate ternary complex and the accuracy-amplifying role of the monitoring bases A1492, A1493 and G530 of the 16S rRNA. We used the GTPase-deficient EF-Tu variant H84A with native GTP, rather than non-cleavable GTP analogues, to trap a near-cognate ternary complex in high-resolution ribosomal complexes of varying codon-recognition accuracy. We found that ribosome complexes trapped by GTPase-deficicent ternary complex due to the presence of EF-TuH84A or non-cleavable GTP analogues have very similar structures. We further discuss speed and accuracy of initial aa-tRNA selection in terms of conformational changes of aa-tRNA and stepwise activation of the monitoring bases at the decoding center of the ribosome.

Figure 1.

(A and C) Cognate tRNA-mRNA interaction in the case of (A) EF-Tu^wt and (C) EF-Tu^H84A bound to the ribosome. (B and D) Orientation of the monitoring bases in the case of (B) EF-Tu^wt and (D) EF-Tu^H84A bound to the ribosome. Binding of both variants of EF-Tu leads to flipping out of the monitoring bases A1492 and A1493, as well as hydrogen bonding between A1492 and G530 (dashed lines). The densities are shown as wire mesh at 2.5 sigma (A and C) around all residues or (B and D) around the monitoring bases only.

Figure 2.

(A) Cognate tRNA-mRNA interaction of the ribosome complex with EF-Tu^H84A•GTP. (B) Orientation of the monitoring bases shows the flipping out of A1492 and A1493, as well as hydrogen bonding between A1492 and G530 (dashed lines). The densities are shown as wire mesh at 2.5 sigma. (C and D) Close-up on the GTPase center of EF-Tu^H84A depicting the (C) successful trapping of GTP and (D) mutation of histidine 84, as well as the structuring of switch II. The densities are shown as wire mesh at 1 sigma around the selected residues.

Figure 3.

(A and B) Near-cognate tRNA-mRNA interaction of the EF-Tu^H84A•GTP bound to the ribosome in the C₄ state. At the mismatch position the mRNA side chain density is reduced indicating increased flexibility. Densities shown at (A) 2.5 sigma and (B) 4 sigma. (C) Orientation of the monitoring bases shows the flipping out of A1492 and A1493, as well as hydrogen bonding between A1492 and G530 (dashed lines). The densities around the monitoring bases only are shown as wire mesh at 2.5 sigma.

Figure 4.

Analysis of the domain movement using domain motion analysis. (A and C) Side view and (B and D) top view of the principal axes of each domain in the open conformation (near-cognate tRNA) and closed conformation (cognate tRNA and C₄), respectively. Axes are shown for EF-Tu (red) and the 16S domains I (blue), III major (green) and III minor (pink). The axes of the reference domain (23S) is highlighted in gray. The small green arrow indicates the axis of rotation going form the near-cognate to the cognate case. Cartoon model representation of the domains are available in Supplementary Figure S3.

Figure 5.

Orientation of the monitoring bases in the case of the near-cognate tRNA complexes (A) C₂, (B) C₃ (C) C₄ as well as (D) the cognate tRNA bound complex. Binding of cognate tRNA, as well as the engaged form of near-cognate tRNA (C₄), leads to flipping out of the monitoring bases A1492 & A1493, as well as hydrogen bonding between A1492 and G530 (dashed lines). In the C₂ complex all monitoring bases are in the ‘off’ state, while in the C₃ complex G530 and A1492 exist in the ‘on’, as well as the ‘off’ state. All densities around the monitoring bases are shown as wire mesh at the indicated sigma levels.

Figure 6.

(A) Overlay of the A/T-site tRNA structures from the complexes C₃ (orange) and C₄ (green) representing the sampling and engaged state respectively. (B) Going from the sampling (orange, C_3a) to the engaged state (green, C₄) of the ribosome, the tRNA moves closer to the P-site tRNA. The bottom overlay compares our study C₃ (orange) and Loveland et al. II_nc (blue).

Figure 7.

Scheme visualizing the ribosome states (blue and yellow ovals) during tRNA selection (black L-shape) by EF-Tu (red circle) in relation to the sarcin-ricin loop (SRL). Comparative naming in other publications is depicted below each state. In the first initial binding step (1) aa-tRNA•EF-Tu•GTP is binding to the open form of the ribosome containing a P-site tRNA (green). During the sampling step (2), the tRNA-mRNA distance is decreases and the A/T-site tRNA undergoes partial bending and (3) induces closure of the ribosome, leading to an engaged state. (4) This is followed by a multi step mechanism leading from GTP hydrolysis to aa-tRNA accomodation via proofreading.