Single-Cell Biophysics: Measurement, Modulation, and Modeling

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Single-Cell Biophysics: Measurement, Modulation, and Modeling Poster Abstracts
49
9-POS Board 5
Structured Mrna Induces Ribosomal Rolling During Frameshifting
Kai-Chun Chang1, Emmanuel Salawu2,3, Yuan-Yu Chang2, Po-Szu Hsieh1, Jin-Der Wen1, Lee-
Wei Yang2,3.
1Institute of Molecular and Cellular Biology, National Taiwan University, Taipei,
Taiwan, 2Institute of Bioinformatics and Structural Biology, National Tsing Hua University,
Hsinchu, Taiwan, 3Bioinformatics Program, Institute of Information Sciences, Academia Sinica,
Taipei, Taiwan.
Programmed ribosomal frameshifting (PRF), where the ribosome slips backwards for one
nucleotide (the “-1” frame), is promoted by a slippery sequence, usually with X-XXY-YYZ
motif, and a road-blocking mRNA structure, such as hairpin or pseudoknot (PK). How the
mRNA structure is unwound during translation and how these elements modify conformational
dynamics of the ribosome to promote PRF remain largely unknown. However, the
conformational dynamics of the ribosome occur at a scale unattainable by MD simulations
(~1.6×104 atoms, 2.4 MDa, at millisecond regime). We circumvent this obstacle by modeling
intrinsic dynamics with coarse-grained anisotropic network model (ANM), followed by
predicting PK-induced perturbed dynamics by linear response theory (LRT). The external
perturbation, which is the tension developed between PK and the ribosome, is obtained from a
series of steps involving cryo-EM fittings and steered molecular dynamics simulations (SMD).
Both ANM and SMD yield results well correlated with X-ray, cryo-EM, single-molecule Förster
resonance energy transfer (smFRET) and mutational studies. Given that the intrinsic dynamics
and perturbation forces are known, LRT predicts global conformational change of the ribosome
upon encountering PK. Surprisingly, the 30S subunit seems to “roll” in a direction orthogonal to
ratcheting, but parallel to the tension of PK. Rolling distorts A/P-tRNA, which is observed by
cryo-EM previously. Subsequent MD simulations further indicate that A/P-tRNA bending
disrupts codon-anticodon interaction. The resulting dissociation of A/P-tRNA and tRNA
slippage are observed in the simulations. Our model, based on first principles, connects and
rationalizes existing experimental data, providing a temporal and spatial description of PRF with
unprecedented mechanistic details.