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至关重要，特别是在肌肉中 和神经元细胞。复杂的核糖体内部结构和延伸因子实现快速和忠实的肽 以GTP能量为代价的延伸周期。然而，这一机制和细胞水平的调节 健康细胞和病变细胞的过程仍然不清楚。例如，由于氨基酸的延伸错误， 错误掺入和移码是神经元变性疾病的根本原因， 心血管疾病、癌症和病毒感染。人核糖体转位酶eEF 2的调控 磷酸化是唯一已知的正常功能修饰，使eEF 2激酶成为一种非常受欢迎的 药物靶点然而，这种修饰如何影响易位尚不清楚。类似地，eEF 1中的突变， 其他延伸因子，导致先天性癫痫和智力残疾，机制尚不清楚。此外，本发明还提供了一种方法， 动态RNA修饰与翻译调节和抗生素抗性有关。我们将解决 这些问题与超分辨力光谱（SURFS），可以直接测量核糖体 在核糖体两侧的mRNA上的脚趾印，并揭示了机械力在其中的作用。 运动这一建议的结果是证明了核糖体的“尺蠖样”易位假说 这是在第一个支持期间提出的模式。它将填补目前的知识空白， 机械力在易位保真度中的作用，揭示相关疾病的新治疗靶点，并产生 生物物理学研究的新工具。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发现的。 它的机制作用在很大程度上是未知的。据我们所知，FIRMS和SURFS是唯一 可以探测核糖体的力和运动的方法。目的是：1）揭示 在动力中风，移码，和动力学中使用延长因子中的致病突变。EF-G EF-Tu在GTP结合口袋和EF-G结构域IV环与tRNA相互作用的突变是 科目2)研究mRNA修饰、密码子重复和抗生素在易位中的作用。之间 核糖体内覆盖的27个mRNA残基，特定位置与rRNA相互作用， 用于阅读帧维护的制动器。这些位置的修饰和抗生素结合是研究的重点。 这个目标。此外，重复mRNA序列的G-四链体如何诱导移码并改变mRNA的表达水平， 动力学将被揭示。3)开发多路时间分辨SURFS。在上一个财政年度，我们 开发了力致剩余磁化光谱（FIRMS），以分辨不同的阅读帧， 确定EF-G及其修改的动力冲程。在第一个供资期即将结束时，次区域资源中心 开发了将声辐射力与FIRMS集成的技术，以获得五倍的力 分辨率在这一目标中，SURFS将实现具有时间分辨率的自动多路复用测量。因此，我们认为， 我们将通过对力和移位步骤的更有效和精确的测量来推进这种技术。

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.