Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

9-POS Board 5 Structured Mrna Induces Ribosomal Rolling During Frameshifting Kai-Chun Chang 1 , Emmanuel Salawu 2,3 , Yuan-Yu Chang 2 , Po-Szu Hsieh 1 , Jin-Der Wen 1 , Lee- Wei Yang 2,3 . 1 Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan, 2 Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan, 3 Bioinformatics 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 (~1.6×10 4 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.

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