A novel force spectroscopy to study the ribosome power strokes and frameshifting

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Dynamic and correct protein synthesis by the ribosome is essential to cell’s normal function, especially in muscle and neuron cells. The intricate ribosome internal structure and elongation factors achieve fast and faithful peptide elongation cycles at the expenses of GTP energy. However, mechanism and cellular level regulations of this process in healthy and diseased cells are still not clear. For example, elongation errors due to amino acid misincorporation and frameshifting are the fundamental causes for neuron degenerative diseases, cardiovascular diseases, cancer, and viral infections. Regulation of the human ribosome translocase eEF2 via phosphorylation is the only known normal functional modification, making the eEF2 kinase an extremely popular drug target. However, how this modification affects the translocation is unclear. Similarly, mutations in eEF1, the other elongation factor, causes congenital epilepsy and intellectual disability with unclear mechanism. In addition, dynamic RNA modifications are connected with translation regulation and antibiotic resistance. We will tackle these problems with super-resolution force spectroscopy (SURFS) that can directly measure the ribosome toeprinting on the mRNA at both sides flanking the ribosome, and reveal the mechanical force’s role in this movement. The outcome of this proposal is to prove the hypothesis of ribosome’s “inchworm-like” translocation model that was proposed during the first supporting period. It will fill the current knowledge gap regarding mechanical force’s role in translocation fidelity, reveal new therapeutic targets for related diseases, and generate a new tool for biophysical research. Our research is unique because force in ribosome translation is only recently confirmed and its mechanistic role is largely unknown. To our best knowledge, FIRMS and SURFS are the only approaches that can probe both force and movement of ribosome. The aims are: 1) reveal the relationship among power stroke, frameshifting, and kinetics using disease-causing mutations in elongation factors. EF-G and EF-Tu’s mutations at the GTP binding pocket and EF-G’s domain IV loops interacting with tRNA are the subjects. 2) investigate the roles of mRNA modifications, codon repeats, and antibiotics in translocation. Among the 27 mRNA residues covered inside the ribosome, specific locations interact with the rRNAs to serve as the brakes for reading frame maintenance. Modifications and antibiotic bindings at these locations are the focus in this aim. In addition, how G-quadruplexes of repeating mRNA sequences induce frameshifting and alter the kinetics will be revealed. 3) develop multiplex time-resolved SURFS. During the previous funding period, we developed force-induced remnant magnetization spectroscopy (FIRMS) to resolve different reading frames and determine the power strokes of EF-G and its modifications. Toward the end of the first funding period, SURFS technique was developed that integrated acoustic radiation force with FIRMS to achieve five-fold better force resolution. In this aim, SURFS will enable automatic multiplexed measurement with time-resolution. Therefore, we will advance this technique with more efficient and precise measurements for force and translocation steps.