The dynamics of SecM-induced translational stalling

Abstract

SecM is an E. coli secretion monitor capable of stalling translation on the prokaryotic ribosome without cofactors. Biochemical and structural studies have demonstrated that the SecM nascent chain interacts with the 50S subunit exit tunnel to inhibit peptide bond formation. However, the timescales and pathways of stalling on an mRNA remain undefined. To provide a dynamic mechanism for stalling, we directly tracked the dynamics of elongation on ribosomes translating the SecM stall sequence (FSTPVWISQAQGIRAGP) using single-molecule fluorescence techniques. Within 1 min, three peptide-ribosome interactions work cooperatively over the last five codons of the SecM sequence, leading to severely impaired elongation rates beginning from the terminal proline and lasting four codons. Our results suggest that stalling is tightly linked to the dynamics of elongation and underscore the roles that the exit tunnel and nascent chain play in controlling fundamental steps in translation.

Figure 1. SecM inhibits elongation rates over specific codons

(A) Cy3B (green) labeled 30S subunits are paired with a non-fluorescent FRET quencher BHQ2 (Black Hole Quencher 2, black)-labeled 50S subunits (Chen et al., 2012b). The ribosome begins elongation in the non-rotated state (low Cy3B intensity). Peptide bond formation rotates the subunits into the rotated (high Cy3B intensity) state. EF-G catalyzed translocation counter-rotates the subunits back to the non-rotated (low Cy3B intensity) state. Each cycle of low-high-low Cy3B intensity represents the translation of one codon. A representative trace from our SecM experiment is shown with state assignment overlaid (Arg15 and Pro18 shown for referecne). (B) Translation efficiency is plotted as the fraction of ribosomes (y-axis) still active over each codon (x-axis) within a 5 minute observation window. Translating the SecM mRNA (n = 347), 93% of the ribosomes do not proceed pass codon 22, with the sharpest drop between codons 18~22. 7% of ribosomes translating past codon 22 are colored light green. (C) The lifetimes of each state, when combined gives the time to translate a codon, are plotted the y-axis against the codon in the A site on the x-axis. The sequence translated is shown with the SecM sequence bolded and the critical amino acids in red. Before codon 15, each codon requires 4~5 s to translate at 2.5 μM total tRNA and 160 nM EF-G. Elongation rates slow down 3~5-fold over codons 16~23 to 15~20 s per codon. The non-rotated lifetimes peak over codons 17~18 and 20~23. The rotated lifetimes remain long between codons 16~22. Lifetimes are fitted to single-exponential distributions and error bars represent s.e.m. See Figure S1 for bulk translation and control experiments.

Figure 2. SecM stalling is not affected by changes in the position of the stall sequence and the concentrations of tRNAs and EF-G

(A) Stalling can be observed near codon 32 when SecM is shifted 10 codons downstream in our intersubunit FRET experiments (n = 242) with 2.5 μM charged total tRNA and 160 nM EF-G. (B) Increases in the non-rotated state lifetimes can be seen around codons 26~33 and increases in the rotated state lifetimes can be seen from codon 25 onwards. (C and D) Doubling charged total tRNA to 5 μM (n = 189), stalling still occurs, but there is a reduction of the non-rotated state lifetimes before codon 16. (E and F) Doubling EF-G to 320 nM, the translation profile (n = 173) remains comparable to SecM, but there is a reduction of the rotated state lifetimes before codon 15. Lifetimes are fitted to single-exponential distributions and error bars represent s.e.m.

Figure 3. tRNA transit experiments show stable tRNA incorporation beyond the terminal proline and reduced elongation rates

(A) Translating with 200 nM of Cy5 labeled tRNA^Phe, accommodation of the tRNA can be tracked through stable Cy5 pulses colocalized to a 30S-Cy3B (Uemura et al., 2010). The tRNA remains on the ribosome for another round of elongation and dissociates once moved into the E site, whose dwell time thus measures the elongation rate. The SecM mRNA contains 32 codons after Phe21 whereas the SecM 3’ truncated mRNA ends exactly at Phe21. The Phe codons (2 and 21) are marked in red. (B) The fraction, normalized to ribosomes showing at least one labeled tRNA binding event, incorporating a tRNA at each Phe codon is shown. At 2.5 μM total tRNA and 160 nM EF-G (n = 220 for normal, n = 151 for truncated), 50% of ribosomes on normal SecM show tRNA binding over Phe21, which is suppressed on the truncated mRNA. (C) Doubling total tRNA to 5 μM and EF-G to 320 nM (n = 382 for normal, n = 288 for truncated) does not change the overall behavior, but increases the fraction of tRNA binding over Phe21. (D) The duration of binding is short over Phe2 but approaches the photobleaching time of the Cy5 dye over Phe21 on the normal SecM mRNA. (E) The time to translate from Phe2 to Phe21 is consistent with intersubunit FRET experiments and decreases when tRNA and EF-G concentrations are doubled. Lifetimes are fitted to single-exponential distributions and error bars represent s.e.m. See Figure S2 for controls and additional tRNA transit experiments.

Figure 4. Proline must be positioned at codon 18 to precipitate stalling

(A) The P18A (Pro18 to Ala) mutant (n = 231), despite retaining Pro19, allows 50% of ribosomes to translate past codon 22. (B) The lifetimes of each state is similar to the wild-type SecM up to codon 17, but the non-rotated state lifetimes quickly recover thereafter and lengthening of the rotated state lifetimes is less. (C) Translating SecM with total tRNA charged with Aze (azetidine-2-carboxylic acid) in place of Pro prevents stalling (n = 293). (D) The time to translate each codon is very similar to the P18A mutant. (E) The P19A mutant (n = 276) behaves exactly like the wild-type sequence, inducing stalling between codons 18~22. (F) The lifetimes per codon profile of P19A is similar to the wild-type SecM. Lifetimes are fitted to single-exponential distributions and error bars represent s.e.m.

Figure 5. Arg15 increases non-rotated state lifetimes and the N-terminal sequence lengthens rotated state lifetimes

(A) The R15A mutant (n = 262) abolishes stalling, allowing 60% of ribosomes to translate past codon 22. (B) No increase in the non-rotated state lifetimes is seen with the R15A mutant and the increase in the rotated state lifetimes is less compared to the wild-type sequence. (C) Deleting the first 9 amino acids (Δ2-10, n = 234) of the SecM sequence abolishes stalling. (D) The lifetimes in each ribosome conformational state remain constant at 4~5 s per codon throughout the truncated sequence. Lifetimes are fitted to single-exponential distributions and error bars represent s.e.m. See Figure S3 for additional experiments on R15 and peptide bond formation.

Figure 6. SecM increases the barrier to translocation but does not hinder EFG binding

(A) Using Cy5 labeled EF-G and Cy3B/BHQ2 intersubunit FRET, EF-G binding attempts to the rotated state leading to a successful translocation can be tracked as a EF-G binding signal concurrent with a high to low Cy3B intensity transition (Chen et al., 2013b). (B) At 2.5 μM charged total tRNA and 160 nM EF-G-Cy5, the average number of EF-G binding attempts increases from 1.5~2.5 to 3.5~4 after codon 15 (n = 91), indicating an increased energy barrier to translocation. Error bars represent standard error. (C) EF-G binding frequency remains constant at 0.4~0.5 s⁻¹, suggesting that EF-G binding to the rotated state is not inhibited. (D) EF-G dwell times on the ribosome remain constant at 0.10~0.15 s. Frequencies and lifetimes are fitted to single-exponential distributions and error bars represent s.e.m.

Figure 7. SecM induced stalling is a dynamic phenomenon that leads to a region of significantly reduced elongation rates

(A) The ribosome translates the SecM sequence normally over the first 13 codons. (B) The peptide then interacts with the constriction point in the exit tunnel, formed by the large subunit proteins L4 and L22. This compacts and increases the mechanical stress on the peptide. The effect propagates downstream via the peptide or the tunnel, leading to reduced translocation rates. (C) Compaction of the peptide positions Arg15 to interact with the exit tunnel entrance to remodel tRNA geometry in the peptidyl transferase center (PTC), slowing down peptide bond formation. (D) This opens a window for Pro18 to lock the SecM peptide in conformations that induces a high level of mechanical stress leading to a heavily remodeled PTC geometry. (E) Elongation rates are greatly slowed over the next 4~5 codons past the terminal proline, leading to stalling that is stable up to an hour.