Biophysical Society Thematic Meeting - June 28-July 1, 2015

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Poster Abstracts

50-POS Board 50 Gymnastics in Translation—Deciphering Ribosomal Frameshifting Dynamics Shannon Yan 1 , Jin-Der Wen 2 , Carlos Bustamante 1,3,4 , Ignacio Tinoco, Jr. 1 . 1 University of California, Berkeley, Berkeley, CA, USA, 2 National Taiwan University, Taipei, Taiwan, 3 University of California, Berkeley, Berkeley, CA, USA, 4 University of California, Berkeley, Berkeley, CA, USA. 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 Escherichia coli dnaX mRNA programs ribosomes to decode not one but two protein products: the 0-frame τ and the -1-frameshifted γ subunits 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)). However, the mechanism, including the timing and location of such a -1-slip, remain unresolved. Here, we determine when within one translation cycle a slippage occurs by following a single ribosome translating a frameshift-programming mRNA held on optical tweezers. In complement, by mass spectroscopy, we survey 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.

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