Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process

Abstract

During the elongation cycle of protein biosynthesis, the specific amino acid coded for by the mRNA is delivered by a complex that is comprised of the cognate aminoacyl-tRNA, elongation factor Tu and GTP. As this ternary complex binds to the ribosome, the anticodon end of the tRNA reaches the decoding center in the 30S subunit. Here we present the cryo- electron microscopy (EM) study of an Escherichia coli 70S ribosome-bound ternary complex stalled with an antibiotic, kirromycin. In the cryo-EM map the anticodon arm of the tRNA presents a new conformation that appears to facilitate the initial codon-anticodon interaction. Furthermore, the elbow region of the tRNA is seen to contact the GTPase-associated center on the 50S subunit of the ribosome, suggesting an active role of the tRNA in the transmission of the signal prompting the GTP hydrolysis upon codon recognition.

Fig. 1. Cryo-EM maps resulting from the classification, and their analysis. Map 1 (A, C and E) and map 2 (B, D and F) are depicted in two orientations related by a 90° rotation around a vertical axis in the plane [(A–D) show the side views, (E) and (F) the top views of the 70S ribosome). Side views are rotated in the plane by 90° from their conventional presentation (see Supplementary figure S1) to maintain continuity in the tRNA positions with (E) and (F)]. In (C)–(F) the ribosomal subunits are represented as semi-transparent surface (blue for the 50S and yellow for the 30S subunit), so that the positions of P- and E-site tRNAs (purple) and the mass attributable to the ternary complex (red) can be seen more clearly. (G) Representation of the difference map (see Materials and methods) calculated for the cryo-EM map 1, shown in green. Lobe of mass labeled with an asterisk is attributed to a conformational change in the L1 region. The ribosome is shown in same orientation as in (C). Landmarks are as follows: CP, central protuberance; Sb, L7/L12 stalk base; sp, spur; b, body; h, head; dc, decoding center; L1, stalk of L1 protein. Labels in tRNA positions: T, T-site within the ternary complex; A, aminoacyl site; P, peptidyl site; E, exit site.

Fig. 2. Comparison of cryo-EM density and X-ray structure of the ternary complex. (A and B) Ternary complex density, isolated from the cryo-EM map: (A) as seen from the solvent side; (B) as seen from the intersubunit space side. (C and D) Equivalent orientations of the X-ray crystal structure from T.aquaticus ternary complex (Nissen et al., 1995), filtered to the resolution of the cryo-EM map. EF-Tu is shown in green, aa-tRNA in yellow. (E and F) Ribbons representation of the fitting of the ternary complex crystal structure into the cryo-EM density. (G–I) EF-Tu from T.aquaticus in the GTP (Kjeldgaard et al., 1993) and GDP (Polekhina et al., 1996) states are shown next to the EF-Tu·GDP·kirromycin inferred from the cryo-EM map at the same resolution.

Fig. 3. Docking of EF-Tu and aa-tRNA into the cryo-EM density of the ternary complex: (A) as seen from the solvent side; (B) as seen from the intersubunit side. (Views are similar, but not identical to those in Figure 2A and B.) Fitted atomic coordinates of E.coli EF-Tu (Song et al., 1999) and Phe-tRNA^Phe [(from the ternary complex of T.aquaticus (Nissen et al., 1995)] are shown inside the semi-transparent cryo-EM density of the ternary complex. The domains of EF-Tu were fitted independently (domain I shown in green, domain II in yellow and domain III pink). The switch-I region within domain I is highlighted in cyan. The dotted line in (A) indicates the place of the kink in the anticodon arm of the tRNA. Orientations of the ribosome are shown as thumbnails on the left. SRL, α-sarcin–ricin loop; L11–rRNA (GAC), protein L11 and the segment of 58 nucleotides of the 23S rRNA, also known as the GTPase-associated center; S12, protein S12 from the small subunit; h5, helix 5 from the 16S rRNA; dc, decoding center in the A-site of the 30S subunit.

Fig. 4. Interaction of EF-Tu and aa-tRNA with the ribosome. (A and B) Ribbons representation of the docked EF-Tu and aa-tRNA within the ternary complex. (C and D) Focus on the interaction between the α-sarcin–ricin loop (SRL) and the effector loop within domain I of EF-Tu (cyan). In (C) the coordinates of the whole ternary complex from T.aquaticus with a GTP analog (Nissen et al., 1995) were used for the fitting, while in (D) the crystal structure of EF-Tu from E.coli bound to GDP (Song et al., 1999) was used. Orientation of the ribosomes for (A) and (B) are shown as thumbnails on the left. Labeling is the same as in Figure 5.

Fig. 5. Interaction in the decoding center and accommodation of the aa-tRNA. (A–C) Semi-transparent representation of the ternary complex density from the cryo-EM map showing the fitted tRNA (gold) and the A-site tRNA (red) with corresponding mRNA codon (Yusupova et al., 2001). (C) A tRNA construct in which the anticodon position, up to the kink, is adopted from (B) and the rest of the tRNA from (A). (D–F) Interaction in the anticodon loop of the tRNA in the decoding site. H69, helix 69 from 23S rRNA; h44, helix 44 from 16S rRNA; h34, helix 34 from 16S rRNA; cd, A-site codon in the mRNA; AC, anticodon loop; S12, protein S12 in the 30S subunit.