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

核糖体的动态和正确的蛋白质合成对细胞的正常功能是必不可少的，特别是在肌肉中 和神经细胞。错综复杂的核糖体内部结构和伸长因子获得快速和忠实的多肽 伸长以消耗GTP能量为代价进行循环。然而，这种作用的机制和细胞水平的调节。 健康和患病细胞中的过程仍然不清楚。例如，由于氨基酸引起的伸长误差 错误整合和移码是神经元退行性疾病的根本原因， 心血管疾病、癌症和病毒感染。人核糖体转位酶eEF2通过 磷酸化是已知的唯一正常的功能修饰，使eEF2激酶成为一种非常受欢迎的 毒品目标。然而，这种修饰如何影响易位尚不清楚。同样，eEF1的突变，即 其他延长因素，导致先天性癫痫和智力残疾，机制不明。此外, 动态的RNA修饰与翻译调节和抗生素耐药性有关。我们会解决的 这些问题与可以直接测量核糖体的超分辨力谱(SURFS)有关 在核糖体两侧的mRNA上留下脚印，并揭示了机械力在其中的作用 有动静。这一提议的结果是证明了核糖体“尺虫状”易位的假说 在第一个支持期内提出的模式。它将填补目前关于以下方面的知识空白 机械力在移位保真度中的作用，揭示相关疾病的新治疗靶点，并产生 生物物理研究的新工具。我们的研究是独一无二的，因为核糖体翻译中的力是最近才出现的 它的机制作用在很大程度上是未知的。据我们所知，公司和冲浪是唯一 既可以探测核糖体的力也可以探测核糖体的运动的方法。目的是：1)揭示两者之间的关系 在力量卒中、移码和利用延长因子的致病突变的动力学之间。EF-G 而EF-Tu在GTP结合口袋和EF-G的结构域IV环与tRNA相互作用的突变是 研究对象。2)研究mRNA修饰、密码子重复和抗生素在易位中的作用。其中 覆盖在核糖体内的27个mRNA残基，特定的位置与rRNA相互作用，作为 用于阅读框架维护的刹车。在这些位置的修饰和抗生素结合是 这个目标。此外，重复的mRNA序列的G-四链如何诱导移码和改变 动力学将会揭晓。3)开发多路时间分辨SURF。在上一个资助期内，我们 开发了力诱导剩余磁化光谱(STRIES)，以解析不同的读数框架和 确定EF-G及其改进型的动力行程。在第一个资助期接近尾声时，SURES 开发了将声辐射力与公司相结合的技术，以实现五倍的更好的力 决议。为了实现这一目标，SURFS将实现具有时间分辨率的自动多路测量。因此， 我们将通过更有效和更精确的力和移位步骤测量来推进这项技术。

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.