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

研究核糖体动力冲程和移码的新型力谱

• 批准号: 9134165
• 负责人: YUHONG WANG
• 金额: $ 26.75万
• 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 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9134165
• 关键词: Antibiotics; Binding; Biological Assay; Codon Nucleotides; Complex; Coupling; DNA; DNA Binding; Data; Disease; Dissociation; Guanosine Triphosphate; HIV; Health; In Situ; Investigation; Kinetics; Lead; Light; Magnetism; Measurement; Measures; Mechanics; Messenger RNA; Methods; Motion; Motor; Motor Neurons; Movement; Peptide Elongation Factor G; Pharmaceutical Preparations; Power stroke; Preclinical Drug Evaluation; Process; Protein Biosynthesis; Proteins; Randomized; Reading; Reading Frames; Research; Research Activity; Ribosomes; Sample Size; Series; Severe Acute Respiratory Syndrome; Signal Transduction; Spectrum Analysis; Surface; Techniques; Therapeutic; Time; Translations; Viral; Virus; Virus Diseases; analog; base; cell growth regulation; cost; design; drug candidate; human disease; magnetic beads; magnetic dipole; mutant; nanodevice; novel; optical traps; prevent; programs; screening; single molecule; stem

 DESCRIPTION (provided by applicant): The ribosome translocation fidelity and the viral programmed frame shifting mechanism are not clear. Solving these questions is fundamentally important and have valuable therapeutic applications to treat viral infections, such as HIV and SARS. The research objective is to apply a novel force spectroscopy (the Force Induced Remnant Magnetization Spectroscopy (FIRMS)) for in situ investigation of the power stroke and frame shifting during the ribosome translocation. This is the only method at present that can measure the EF-G mechanical force being involved in the ribosome translocation. In addition, different ribosome subpopulations are selectively detached from the surface with different centrifugal forces. Therefore, FIRMS can detect inhomogeneous subpopulations without the ensemble average effect, which is difficult to achieve with either optical trap techniques or ensemble methods. The currently used optical trap method is limited by the weaker mRNA-ribosome interactions, which will lead to the dissociation of the mRNA-ribosome complex before the power stroke can be measured; the small sample sizes and broad distribution of data prevent this single molecule method to fully apply its potential to distinguish inhomogeneous subpopulations. In FIRMS, the ribosome complex is tethered with a magnetic micro-bead at one terminus of the mRNA, while the other terminus hybridizes with a surface-bound DNA. The dissociation of the mRNA-DNA duplex by the power stroke or an external mechanical force leads to randomization of the magnetic dipoles of the micro-beads, which results in a decrease in the magnetic signal detected by an atomic magnetometer. The measurements are twofold. One is to use a series of duplexes as internal force references, whose binding forces can be precisely determined by FIRMS, to noninvasively measure the mechanical force generated by motor proteins. The other is to determine the ribosome-uncovered-mRNA sequence to reveal the ribosome movement with single base accuracy by measuring the binding force between the mRNA-DNA duplex. The specific aims are: 1. Reveal the correlation between the EF-G power stroke and translocation fidelity; 2. Develop an in situ frame shifting assay to reveal the step-by step mechanism of viral "-1" frame shifting mechanism. The ribosome is a major junction point of the cellular regulation network. Revealing the EF-G power stroke will shed light on the mechanism of the natural chemomechanical coupling in motor proteins and help to design manmade nano- devices for higher energy efficiency. The frame shifting assay will provide a platform to screen drug-like molecules to treat the viral infections targeting the frame shifting motifs. In the long term, this method can be used to study a broad range of motor proteins, many of which are closely related to human diseases such as motor neuron degeneracy diseases.

 描述(申请人提供)：核糖体移位的保真度和病毒编程移框机制尚不清楚。解决这些问题从根本上说是重要的，并对治疗艾滋病毒和SARS等病毒感染具有宝贵的治疗应用价值。本研究的目的是应用一种新的力谱(力诱导剩余磁化谱)来原位研究核糖体易位过程中的功率行程和框架移位。这是目前唯一能测量参与核糖体转位的EF-G机械力的方法。此外，不同的核糖体亚群在不同的离心力下被选择性地从表面分离。因此，公司可以在没有系综平均效应的情况下检测不均匀的子总体，这是用光学陷阱技术或系综方法都难以实现的。目前使用的光学捕获方法受到较弱的mRNA-核糖体相互作用的限制，这将导致在功率脉冲被测量之前mRNA-核糖体复合体的解离；小样本量和广泛的数据分布阻碍了这种单分子方法在区分不同亚群方面的潜力。在公司中，核糖体复合体在mRNA的一端与磁性微珠相连，而另一端与表面结合的DNA杂交。微珠的磁偶极子在动力或机械外力作用下发生解离，导致微珠磁偶极子的随机化，从而导致原子磁强计探测到的磁信号减弱。测量结果是双重的。一种是使用一系列双链作为内力参考，其结合力可以由公司精确确定，以非侵入性地测量马达蛋白质产生的机械力。另一种是通过测量核糖体-DNA双链之间的结合力来确定核糖体未被覆盖的mRNA序列，以单碱基的精度揭示核糖体的运动。具体目的是：1.揭示EF-G功率卒中与移位保真度之间的关系；2.建立原位移帧实验来揭示移位 病毒“-1”移框机构的步进机构。核糖体是细胞调控网络的主要连接点。EF-G功率冲程的揭示将有助于揭示马达蛋白质中自然化学机械耦合的机制，并有助于设计出具有更高能效的人造纳米器件。移框试验将为筛选类药物分子提供一个平台，以治疗针对移框基序的病毒感染。从长远来看，这种方法可以用于研究广泛的运动蛋白，其中许多与运动神经元退行性疾病等人类疾病密切相关。