Gymnastics in Translation—Deciphering Ribosomal Frameshifting Dynamics

The genetic content of a messenger RNA (mRNA) can be recoded and hence expanded when the translating ribosomes are programmed to switch reading frames. For instance, the dnaX gene mRNA from Escherichia coli programs ribosomes to synthesize not one but two protein products: the 0-frame encoded τ subunit and the -1-frameshifted γ subunit for DNA polymerase III. It was thought that the latter product is translated via a probabilistic -1-nucleotide slip midway during translation across a slippery sequence, AAAAAAG. mRNA structural barriers flanking the slippery sequence—i.e. a Shine-Dalgarno:anti-Shine-Dalgarno mini-helix and a stable hairpin—can further “actuate” the ribosome to frameshift with an efficiency as high as 80% (= (γ/(γ+τ))×100%). However, the mechanism, including the timing and location of such a -1-slip, remain unresolved.In my PhD work presented here, I attempted to determine when within one translation cycle a slippage occurs by following a single ribosome translating a frameshift-programming mRNA held on optical tweezers. To complement the translation dynamics observed in real time, by mass spectrometry, I surveyed the entire pool of synthesized polypeptides to identify on which codon the ribosome slipped.Mass spectrometry of translated products shows that ribosomes enter the -1 frame from not one specific codon but various codons along the slippery sequence and slip by not just -1 but also -4 or +2 nucleotides. Coincidentally, single-ribosome translation trajectories detect distinctive codon-scale fluctuations in ribosome-mRNA displacement across the slippery sequence, representing multiple ribosomal translocation attempts during frameshifting. Flanking mRNA structural barriers mechanically stimulate the ribosome to undergo back-and-forth translocation excursions, thereby permitting the ribosome to explore alternative reading frames. Both experiments reveal aborted translation around mutant slippery sequences, indicating that subsequent fidelity checks on newly adopted codon position base pairings lead to either resumed translation or early termination. What has then emerged from our results is a versatile ribosomal frameshifting scheme during mRNA translocation, mediating broad branching of frameshift pathways.Occurrence of the unsuccessful frameshifting thus prematurely terminated events had been previously overlooked and now provides a proper metric accounting for every frameshifting attempt. These events not only represent a biological context where translation quality critically relies on retrospective fidelity checks conducted by the ribosome. They result in stalled ribosomes on the mRNA—which are known targets constantly under cellular surveillance—and hence implicate canonical signaling critical for cellular regulation network