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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.