Crystal structure of 70S ribosome with both cognate tRNAs in the E and P sites representing an authentic elongation complex

Abstract

During the translation cycle, a cognate deacylated tRNA can only move together with the codon into the E site. We here present the first structure of a cognate tRNA bound to the ribosomal E site resulting from translocation by EF-G, in which an entire L1 stalk (L1 protein and L1 rRNA) interacts with E-site tRNA (E-tRNA), representing an authentic ribosome elongation complex. Our results revealed that the Watson-Crick base pairing is formed at the first and second codon-anticodon positions in the E site in the ribosome elongation complex, whereas the codon-anticodon interaction in the third position is indirect. Analysis of the observed conformations of mRNA and E-tRNA suggests that the ribosome intrinsically has the potential to form codon-anticodon interaction in the E site, independently of the mRNA configuration. We also present a detailed description of the biologically relevant position of the entire L1 stalk and its interacting cognate E-tRNA, which provides a better understanding of the structural basis for translation elongation. Furthermore, to gain insight into translocation, we report the positioning of protein L6 contacting EF-G, as well as the conformational change of the C-terminal tail of protein S13 in the decoding center.

Figure 1. Overall structure of 70S ribosome with a cognate tRNA and codon-anticodon interaction in the E site.

(a). Overall structure of two cognate tRNAs (P and E sites) bound to 70S ribosome complexed with EF-G representing an authentic posttranslocational state. EF-G, colored violet (same as below), is represented as surface model. Three ribosomal proteins, L1, L6, and S13, that will be described in the text, colored green, black (surface show), and red, respectively, are labeled in the overall structure. The codon-anticodon in E site is indicated by dashed rectangle. (b). Unbiased difference Fourier electron density map displayed at 1.2σ with refined E-site mRNA and tRNA. Based on the map, one water molecule was located and shown as firebrick sphere. (c). Interactions of mRNA and tRNA in both P and E sites. The dashed lines indicate hydrogen bonds, and W represents one water molecule.

Figure 2. Close-up view of ribosomal elements around E codon and the conformational change.

RNA helices are numbered with the standard Brimacombe nomenclature, prefixed by H for 23S rRNA and h for 16S rRNA, and RNA residues are numbered with the E. coli sequence throughout this paper. Except for non-cognate tRNA and mRNA colored grey that are taken from our previous structure (PDB 2WRI), other components, 16S rRNA (colored cyan), S7 (colored orange), tRNA and mRNA are presented in the present ribosome complex. The E codon and the immediately upstream nucleotide are shown as stick, are labeled by −4 to −1 based on the position related to the first nucleotide A (position +1) in the original P codon AUG of the mRNA Z4C. Upon establishing codon-anticodon interactions, ASL shifts by ∼5 Å, apart from h28 of 16S rRNA, resulting in disruption of ASL interaction with 16S rRNA, indicated by double-headed arrow. Both tRNA anticodons in the two structures points towards mRNA codon, therefore are not shown.

Figure 3. Interactions of L1 protein, L1 rRNA and the cognate E-tRNA.

(a). A complete model of entire L1 stalk interacting with E-tRNA in 70S. The newly built H78 is colored blue. (b). Interactions of domain I of L1 with 23S rRNA. (c). The detailed interactions among L1 stalk (L1 protein and L1 rRNA) with E-tRNA.

Figure 4. Structural comparison of EF-G with EF-Tu bound a distorted A-tRNA in the ribosome and conformational change of the C-terminal tail of ribosomal protein S13.

The bound GDP in EF-G was shown as yellow surface model, indicated by arrow. The head, body, and shoulder domains of 16S rRNA (cyan surface model) are labeled. (a). Superposition of EF-Tu to EF-G in ribosome by fitting 23S rRNA. EF-G and EF-Tu complexed with tRNA are held in the same pocket, surrounded by 23S rRNA (sarcin-ricin loop SRL, L11 rRNA including H43 and H44, intersubunit bridge B2a H69), and 16S rRNA spanning both head (h31) and body (h18). (b). Conformational change of C-tail of protein S13. Protein S13 in the present structure and EF-Tu bound to ribosome are colored red and grey.

Figure 5. The positioning of protein L6 and its interaction with EF-G.

(a). L6 and elements of EF-G in the vicinity of the L11 rRNA region (H43–44) and the SRL. L6 was represented as surface model in grey. (b). Overlarge view of detailed interactions of L6 and EF-G involving L11 rRNA and SRL.