EF-G–induced ribosome sliding along the noncoding mRNA

Figures

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  Fig. 1. EF-G initiates translational bypassing. (A) Schematic of the gene 60 mRNA. ORF1 (light green) spans codons 1 to 46 up to the Gly take-off codon, GGA (green), followed by a UAG stop codon (red). ORF2 (blue) begins with a Leu codon UUA after the landing codon (green) (1, 3). (B) The translation products of gene 60 visualized using a fluorescent reporter, BODIPY FL, incorporated cotranslationally at the N terminus of the nascent peptide (3). mRNA constructs that code for ORF1 alone (stop), the full-length protein consisting of ORF1+ORF2 separated by a noncoding gap (WT), or the full-length protein synthesized as a single ORF1+ORF2 polypeptide without the gap (Δgap). The short peptide corresponds to amino acids 1 to 46 (stop); bypassing results in a 160 amino acids long product that includes the second ORF (byp). (C) Temperature dependence of bypassing. (D) Identification of the component that initiates bypassing. Take-off complexes were prepared and purified from translational components at 10°C and transferred to 37°C (take-off). In all other experiments, EF-Tu–GTP–[14C]Leu-tRNA was added in the absence of EF-G (−EF-G) or in the presence of EF-G(WT), EF-G with mutations in domain 4…

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  Fig. 2. The role of mRNA-encoded elements in initiating bypassing. (A) mRNA elements required for bypassing. The structure of the nascent peptide in the exit tunnel (left top inset) is from a cryo-EM structure (6). PTC, peptidyl transferase center of the ribosome. The schematic of the α-helical nascent peptide and the amino acid sequence indicated below show the positions (red) that affect bypassing (3). Circles in the 5′SL indicate mutations introduced to disrupt (4M) or to restore (4MC), the putative secondary structure of the 5′SL (3). The mutated part of the take-off SL is highlighted in pink; the mutations in the loop are shown below the mRNA sequence. The cryo-EM structure of the folded A-site SL (purple) and the P-site tRNA (green) within the ribosome (blue) (6) is shown in the right inset. (B) Effect of mutations in the apical part of the take-off SL on bypassing [nomenclature taken from (1)]. (C) Bypassing efficiency at predicted folding energies of the take-off SL variants. (D) Temperature dependence of bypassing. (E) Effect of mutations in the take-off SL, 5′SL, and the nascent peptide (Y16H, Y16H/V22D, and KKYK mutated to EEHE) on the temperature dependence of bypassing.…

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  Fig. 3. Ribosomal protein S12 and EF-G tune the temperature dependence. (A) Schematic of mutations in S12 and EF-G. Superposition of the structures of a pretranslocation complex with tRNAs in the A and P sites (the tRNAs are red and green, respectively) and EF-G (yellow) (38) and of the take-off complex with the P-site peptidyl-tRNA (light green) and the SL in the A-site (purple) (6). The position of the A-site SL in the bypassing complex overlaps with the A-site tRNA anticodon domain. Positions of point mutations in EF-G and S12 (dark blue) are indicated. (B) The temperatures Tb at which bypassing is half-maximal is shown for the combinations of WT and mutant S12 and EF-G. Error bars show the SD for n = 3 replicates. **P < 0.005 and ***P < 0.0005 by Student’s two-tailed unpaired t-test.

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  Fig. 4. Ribosome conformation during bypassing visualized by smFRET. (A and B) FRET distribution histogram for the purified take-off complexes at 4° to 10°C and 22°C, respectively. Black lines indicate the positions of main ribosome populations with FRET efficiencies of 0.3, 0.5, and 0.7 (for statistics, see table S1). Tim is the imaging temperature. (C) Landing complex obtained by incubation of the take-off complex with EF-G (1 μM) and with GTP for 1 min at 37°C and imaged at 22°C. (D) Ribosome conformation after addition of EF-G–GTP and EF-Tu–GTP–Leu-tRNA at 37°C; imaging was at 22°C. (E) Time-resolved smFRET recorded upon addition of EF-G (10 nM) with GTP to the imaging buffer. Time courses were synchronized at the first observed transition. Contour plot represents transitions from FRET states of 0.3 to 0.5; other types of transitions were not observed. (F) Distribution of hyper-rotated, rotated, and nonrotated conformations in different complexes normalized to the activity of the take-off complex. (G and H) FRET distribution histograms for the SecM-stalled ribosome complexes at 4° to 10°C and 22°C.