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

用于研究核糖体动力冲程和移码的新型力谱

• 批准号: 10693913
• 负责人: YUHONG WANG
• 金额: $ 29.45万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10693913
• 关键词: Acoustics; Affect; Amino Acids; Antibiotic Resistance; Antibiotics; Binding; Biological Assay; CAG repeat; Cardiovascular Diseases; Cells; Child; Codon Nucleotides; Complement; Congenital Epilepsy; DNA Tumor Viruses; Degenerative Disorder; Detection; Disease; Drug Targeting; Elongation Factor; Epilepsy; Exons; Funding; G-Quartets; GTP Binding; Generations; Grant; Guanosine Triphosphate; Health; Homologous Gene; Human; Huntington Disease; Huntington gene; Intellectual functioning disability; Kinetics; Knowledge; Legal patent; Location; Maintenance; Malignant Neoplasms; Manuscripts; Measurement; Measures; Messenger RNA; Methods; Modeling; Modification; Movement; Muscle; Mutation; Neurons; Nucleotides; Outcome; Paper; Peptide Elongation Factor G; Peptides; Phase; Phosphorylation; Phosphotransferases; Play; Positioning Attribute; Power stroke; Process; Protein Biosynthesis; RNA; Radiation; Reading Frames; Regulation; Research; Resistance; Resolution; Ribosomal RNA; Ribosomes; Role; Sampling; Scheme; Side; Spectrum Analysis; Structure; Techniques; Time; Transfer RNA; Translational Regulation; Translations; Virus Diseases; base; biophysical tools; design; disease-causing mutation; human disease; improved; insight; mechanical force; multiplex detection; mutant; new technology; new therapeutic target; novel; sensor; therapeutic target; translocase; ultra high resolution

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.

核糖体动态且正确的蛋白质合成对于细胞的正常功能至关重要，尤其是在肌肉中 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、忠实的肽 以 GTP 能量为代价的伸长周期。然而，这种机制和细胞水平的调节 健康细胞和患病细胞中的这一过程仍不清楚。例如，由于氨基酸导致的伸长误差 错误掺入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 的调节 磷酸化是唯一已知的正常功能修饰，使得 eEF2 激酶成为非常受欢迎的 药物靶点。然而，这种修饰如何影响易位尚不清楚。同样，eEF1 的突变 其他伸长因素，导致先天性癫痫和智力障碍，但机制尚不清楚。此外， 动态 RNA 修饰与翻译调节和抗生素耐药性有关。我们将解决 可以直接测量核糖体的超分辨率力谱（SURFS）解决这些问题 核糖体两侧 mRNA 上的脚趾印记，揭示了机械力在此过程中的作用 移动。该提案的结果是证明核糖体“类似尺蠖”易位的假设 在第一个支持期间提出的模型。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 生物物理研究的新工具。我们的研究是独一无二的，因为核糖体翻译力最近才出现 已得到证实，但其机制作用在很大程度上尚不清楚。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）揭示关系 在动力冲程、移码和动力学中使用伸长因子中的致病突变。 EF-G EF-Tu 在 GTP 结合口袋处的突变以及 EF-G 与 tRNA 相互作用的结构域 IV 环是 科目。 2) 研究 mRNA 修饰、密码子重复和抗生素在易位中的作用。之中 核糖体内覆盖的 27 个 mRNA 残基，特定位置与 rRNA 相互作用，作为 用于阅读架维护的制动器。这些位置的修饰和抗生素结合是重点 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 动力学将被揭示。 3）开发多重时间分辨SURFS。在上一个资助期间，我们 开发了力诱导剩磁光谱（FIRMS）来解析不同的阅读框架和 确定 EF-G 及其改进型的动力冲程。在第一个资助期结束时，SURFS 开发了将声辐射力与 FIRMS 相结合的技术，以实现五倍更好的力 解决。为此，SURFS 将实现具有时间分辨率的自动多路测量。所以， 我们将通过更有效、更精确地测量力和易位步骤来推进这项技术。