Structural basis for mRNA and tRNA positioning on the ribosome

Abstract

Protein synthesis requires the accurate positioning of mRNA and tRNA in the peptidyl-tRNA site of the ribosome. Here we describe x-ray crystal structures of the intact bacterial ribosome from Escherichia coli in a complex with mRNA and the anticodon stem-loop of P-site tRNA. At 3.5-A resolution, these structures reveal rearrangements in the intact ribosome that clamp P-site tRNA and mRNA on the small ribosomal subunit. Binding of the anticodon stem-loop of P-site tRNA to the ribosome is sufficient to lock the head of the small ribosomal subunit in a single conformation, thereby preventing movement of mRNA and tRNA before mRNA decoding.

Fig. 1.

Overall structure of intact E. coli 70S ribosome in a complex with mRNA and P-site ASL. (A) Secondary structures of the ASL and mRNA–ASL codon–anticodon base pairing. Numbering of mRNA and ASL nucleotides is indicated. (B) Anisotropic diffraction from crystals of the 70S ribosome complexed with a P-site ASL and mRNA. Completeness of the data as a function of resolution is plotted on the right axis, and the mean signal-to-noise of diffraction intensities, or I/σ(I), is plotted on the left axis. The dashed line represents a mean I/σ(I) of 2. (C) Conformational rearrangement of the 70S structure upon binding of mRNA and P-site ASL. Rearrangements of the 30S head positions in the apo-70S ribosome structures are indicated by difference vectors between phosphorous atoms (light blue) and Cα atoms (dark blue). In the ribosome complex, the ASL and mRNA are shown as space-filling models, and the 5′ to 3′ direction of mRNA is also indicated. Letters indicate the approximate alignments of the aminoacyl (A), peptidyl (P), and exit (E) tRNA binding sites at the subunit interface. Domains of the ribosome are labeled for the 30S head (Head), and 50S central protuberance (CP), as are ribosomal proteins in the small (S) and large (L) subunits.

Fig. 2.

Interactions between the ribosome and mRNA in the P site. (A) Hydrogen bonds to the phosphates of nucleotides +1 and +3 of mRNA shown from the perspective of the 30S head. The position of G1401 and a fully hydrated Mg²⁺ have been removed for clarity. (B) Coordination of a fully hydrated Mg²⁺ to 16S rRNA and the backbone of mRNA, shown from the perspective of the subunit body. (C) View of (F_obs − F_calc) difference electron density in the electronegative pocket between the backbone of P-site mRNA and helix-44 nucleotides 1494–1498 in 16S rRNA. The position of A1493 is already adjusted to fit the electron density. (D) Nucleotides +4 through +6 of the mRNA, along with two Mg²⁺ ions, modeled into the electron density in C followed by refinement.

Fig. 3.

Ribosome interactions with the P-site ASL. (A) Closing of the P-site cleft when compared with the 30S ribosome structures (9). Changes in the distance between C1400 and G966 and between G966 and ASL are indicated by arrows and distances. The position of the very C terminus of protein S9 is indicated. The view is from the aminoacyl-tRNA site in the small subunit. (B) Stereoview of the averaged (3F_obs − 2F_calc) difference electron density for the 30S contacts to the ASL shown in A. Electron density for mRNA nucleotides +4 through +6 has been removed for clarity. The density indicated by an asterisk is disconnected from that for protein S9 and therefore has not been assigned. (C) Minor groove interactions among G1338, A1339, and the P-site ASL. The view is from the right in A, i.e., from the perspective of the 50S subunit.

Fig. 4.

Closing of the 30S head domain in the 70S ribosome when compared with the 30S subunit structure. The structure of Protein Data Bank entry 1J5E was used for comparisons (9). The direction of closing of the small subunit head is indicated by an arrow; i.e., the axis of rotation is perpendicular to the plane of the image. The P-site ASL (green) and mRNA (orange) are also shown. The position of full-length tRNA in the P site as modeled based on the ASL positions is shown in outline form, with the arrow indicating the change in direction in going from the 30S to 70S structures.