Molecular architecture and diversity of StopGo/2A translational recoding

Figures

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  Fig. 1.. Cryo-EM structure of the F2A-StopGo complex. (A) Schematic of the F2A-PGX mRNA (Top; X = stop codon) and complex preparation strategy (Bottom). (B) Anti-FLAG Western blot of in vitro translation reactions of the F2A-PGX mRNA in absence/presence of eRF1-AAQ. The free and tRNA-bound F2A-NC species are labeled. (C and D) Cryo-EM maps of the complete F2A-StopGo complex (C), or E- and P-site tRNAs, F2A-NC, and eRF1-AAQ, colored based on local resolution (D). (E and F) Map and model of P-site tRNAGly, the attached F2A-NC, and eRF1-AAQ in the PTC region (E) or the mRNA (F). The disordered region around the eRF1-AAQ motif (E) and the A, P, and E-sites (F) are highlighted.

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  Fig. 2.. Interactions of the F2A-NC and the ribosomal peptide exit tunnel. (A) Cryo-EM density and model of tRNAGly-CCA end and attached F2A-NC. (B) Path of the F2A-NC in the ribosomal peptide exit tunnel. Residues constituting a hydrophobic cluster are highlighted. (C) Polar interactions between F2A-NC side chains and 28S rRNA residues. Homologous rRNA nucleotides of E. coli are given in parentheses; the orientation relative to (B) is indicated. (D) Autoradiograph of SDS-PAGE gel showing StopGo activity assay of F2A single mutations using a dual reporter construct (Top). The calculated StopGo efficiency (%) and SD (±%) are given below (n = 3).

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  Fig. 3.. Inactivation of the PTC in the F2A-StopGo complex. (A–C) Superposition of the models of the F2A-StopGo complex and a canonical termination complex in the pre-release state, trapped with eRF1-AAQ (PDB: 5LZV), showing the P-tRNA/NC (A), close-up view of the CCA end/NC C-terminus (B), or PTC (C). Models are colored as indicated; structural changes to the positioning of the P-tRNA, trajectory of the NC or rRNA residues of the PTC imposed by the F2A-NC are indicated by arrows. E. coli numbering of rRNA residues is given in parentheses. (D) Positioning of the P- and (modeled) A-site tRNAs within the PTC of the F2A-StopGo complex (Left) or a bacterial PRE elongation complex in the pre-transpeptidation state (Right, PDB: 8CVJ). The A-tRNAPro was modeled based on the A-site tRNA of PDB model 6Y0G. The distances between the ester bond and amino group of the A-site aminoacyl-tRNAs are indicated. (E) Autoradiograph showing StopGo activity assay of amino acid substitutions of F2A P19 using a dual reporter construct (Top). The calculated StopGo efficiency (%) and SD (±%) are given below (n = 3).

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  Fig. 4.. Bioinformatic identification of StopGo core octapeptide variants. Starting with the NCBI, Edgar, Neri, Zayed, Wolf, and Wu sequences, ORFs were identified and clustered with CD-HIT using a 70% amino acid identity threshold. All octapeptides differing from D(V/I)ExNPGP by at most one position were identified, yielding 908 exact matches and 1,557 inexact matches. (A) At each site, the proportion of the 2,465 octapeptides exhibiting each of the 20 amino acids was quantified. (B) Same as panel A but with the y-axis enlarged 50× (4× for p15) to facilitate viewing of rare variants (the bars corresponding to prevalent variants extend beyond the top of the graphs). (C) Same as panel B but with amino acid proportions inversely scaled by overall amino acid frequencies in the input database. Blue bars at far right show the expected total number of octapeptides (i.e., summed over all possible amino acid substitutions) differing from D(V/I)ExNPGP at only the given position, in a randomized database with the same size and amino acid frequencies as the input database; f.p. denotes false positives. (D and E) StopGo sequences containing rare variants in the core octamer (D), used for the StopGo…

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  Fig. 5.. Identification of distinct motif classes upstream of the StopGo core octapeptide and host distribution of core octapeptides and upstream hexapeptide motifs. (A) Weblogo produced from 1,900 representative StopGo sequences. The depicted region covers from 14 positions upstream of the octapeptide to 8 positions downstream. The core octapeptide is highlighted (red dashed box). (B) Weblogos for sequences identified by FIMO from among the 1,900 representative sequences as matching each of the six MEME-defined hexapeptide motifs. (C) StopGo sequences containing examples of the upstream motifs identified in (B), and mutants of the most characteristic positions. (D) StopGo activity assay of the sequences in (C), based on a dual reporter construct (Top). The calculated StopGo efficiency (%) and SD (±%) are given below (n = 3). (E) Representation of host phyla in the input sequence database (total number of amino acids; Left) and among the identified (D/G/C/N)(V/I)ExNPGP octapeptides (Right) for NCBI RNA virus sequences with designated host species. (F) Representation of hexapeptide motifs 1 to 6 (M1 to M6), besides unclassified hexapeptides (Un.), upstream of (D/G/C/N)(V/I)ExNPGP…

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  Fig. 6.. Proposed model for the StopGo mechanism. During translation of a StopGo sequence, the StopGo-NC (F2A in this study) interacts with the nascent peptide exit tunnel of the elongating ribosome, impeding ribosomal processivity (Bottom Left). A conformational change in the PTC induced by the StopGo-NC stabilizes the P-tRNA CCA end in an inactive state, which inhibits peptidyl transfer (Bottom and Middle) but may pre-expose the tRNA:StopGo-NC ester bond for hydrolysis (Bottom Right). After NC release, translation continues with the downstream product starting with P19. In the Bottom Middle panel, the F2A-NC was modeled in the canonical conformation based on PDB ID 7O80 (transparent P-tRNA/NC); the A-tRNAPro was modeled based on PDB ID 6Y0G.