High-resolution structure of the Escherichia coli ribosome

Abstract

Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation have remained poorly understood. Moreover, the functions of modifications to ribosomal RNA and ribosomal proteins have also been unclear. Here we present the structure of the Escherichia coli 70S ribosome at 2.4-Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and it suggests how solvation contributes to ribosome integrity and function as well as how the conformation of ribosomal protein uS12 aids in mRNA decoding. This structure helps to explain the phylogenetic conservation of key elements of the ribosome, including post-transcriptional and post-translational modifications, and should serve as a basis for future antibiotic development.

Figure 1

E. coli 70S ribosome I in an intermediate rotated state. (a) Ribosomal subunits of ribosome I colored by atomic displacement factor (ADP) from 20 to 150 Ų. The views are from the perspective of the subunit interface. Features in the 50S subunit include the central protuberance (CP), L1 arm (L1), protein L9 (L9), L7–L12 region (L12), A-site finger (ASF) and the GTPase center (G). In the 30S subunit, these include the head (H), body (B), and platform (PL). (b) Single-molecule imaging of the modulation of ribosome dynamics by magnesium ions. Occupancy of unrotated (red) and rotated (black) states of the ribosome as measured by smFRET between fluorophores on uS13 and uL1, shown with standard deviations of three technical replicates (Online methods).

Figure 2

Solvation at the ribosomal subunit interface and in the nascent peptide exit tunnel. (a–c) Water molecules that bridge the 16S (blue) and 23S (grey) rRNA in bridge B3 of ribosome I. 2′-hydroxyls involved in the water interactions are marked with asterisks. The location of bridge B3 is indicated in the inset. The feature enhanced maps are contoured at 2.5 standard deviations from the mean. (d) Solvation at the entrance of the nascent peptide exit tunnel. Labeled residues of 23S rRNA are shown in orange. The feature enhanced map is contoured at 2.0 standard deviations from the mean.

Figure 3

Posttranscriptional and posttranslational modifications in functional centers of the ribosome. (a) View of the peptidyl transferase center (PTC) with C75 of the P-site tRNA (orange) modeled from PDB ID 3I8H and 3I8I. Hydroxyarginine 81 (magenta) is in protein uL16 and Gm2251, dihydrouridine 2449 and m²G2445 are in 23S rRNA (grey). (b) Hydroxylation of arginine 81 of uL16 at the Cβ position is in the R configuration. The feature enhanced map is contoured at 2.5 standard deviations from the mean. (c) Dihydrouridine 2449 and A2448 assume a C2′-endo sugar pucker. Feature-enhanced electron density map shown as in (b). (d) Geometry of protein uS12 (cyan) near the mRNA decoding center. Proline 45 of uS12 in the cis-peptide conformation would be positioned less than 4 Å away from the 3rd nucleotide in the mRNA A-site codon (green). mRNA is modeled from PDB ID 4QCQ. (e) Pro45 in uS12 with a cis-peptide bond and β-methylthioaspartate in the R configuration at position 89 shown with feature enhanced maps contoured at 2.5 and 2.2 standard deviations from the mean, respectively. (f) Model of mRNA (PDB ID: 4QCQ) superimposed on the E. coli high-resolution structure, in which protein uS12 in the decoding site is modeled with a trans-3-hydroxy-proline at position 45. The 3-hydroxy group of Pro45 would be in hydrogen bonding distance of the O3′ of the 3rd position of the A-site codon.

Figure 4

Pseudouridines and syn-pyrimidines in the ribosome. (a) Pseudouridine with a major groove water molecule. Shown is the N1 imino group of ψ955 in 23S rRNA hydrogen bonding to a water molecule that in turn hydrogen bonds to the non-bridging phosphate oxygen of ψ955 and G954. The feature enhanced map is contoured at 2.5 standard deviations from the mean. (b–d) Examples of syn-pyrimidines in the ribosome. Nucleotide U960 in a syn conformation stabilizes a triloop capping helix h31 in 16S rRNA by forming a reverse U-U base pair with U956. (c) The feature enhanced map for the U960–U956 base pair is contoured at 2.5 standard deviations from the mean. (d) Nucleotide U1779 in 23S rRNA adopts a syn conformation to form an unusual reverse Hoogsteen A–U base pair with A1784, allowing the N6 exocyclic amino group of A1784 to hydrogen bond with a non-bridging phosphate oxygen of U1779. The feature-enhanced electron density map is contoured at 2.5 standard deviations from the mean. Hydrogen bonds are indicated by red dashed lines. The hydrogen bond marked with an asterisk is 3.3 Å long.