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

 描述（申请人提供）：核糖体易位保真度和病毒程序性移码机制尚不清楚。解决这些问题是非常重要的，并有价值的治疗应用，以治疗病毒感染，如艾滋病毒和SARS。研究目标是应用一种新型的力谱（力诱导剩余磁化谱（FIRMS））来原位研究核糖体移位过程中的动力行程和框架移位。这是目前唯一可以测量参与核糖体易位的EF-G机械力的方法。此外，不同的核糖体亚群以不同的离心力选择性地从表面脱离。因此，FIRMS可以检测不均匀的亚群没有系综平均效应，这是很难实现的光学陷阱技术或系综方法。目前使用的光阱方法受到较弱的mRNA-核糖体相互作用的限制，这将导致在可以测量动力冲程之前mRNA-核糖体复合物的解离;小样本量和广泛的数据分布阻止了这种单分子方法充分发挥其区分不均匀亚群的潜力。在FIRMS中，核糖体复合物在mRNA的一端与磁性微珠栓系，而另一端与表面结合的DNA杂交。mRNA-DNA双链体通过动力冲程或外部机械力的解离导致微珠的磁偶极子的随机化，这导致原子磁力计检测到的磁信号的降低。测量是双重的。一种是使用一系列双链体作为内力参考，其结合力可以通过FIRMS精确测定，以非侵入性地测量马达蛋白产生的机械力。另一种方法是通过测定核糖体-未覆盖的-mRNA序列，通过测量mRNA-DNA双链体之间的结合力，以单碱基精度揭示核糖体的运动。具体目标是：1.揭示EF-G动力性卒中与易位保真度的相关性;开发原位移码分析，以揭示 病毒“-1”移码机制的阶梯机制。核糖体是细胞调控网络的主要连接点。揭示EF-G动力冲程将揭示马达蛋白中天然化学机械耦合的机制，并有助于设计更高能效的人造纳米器件。移码试验将为筛选靶向移码基序的药物样分子提供一个平台。从长远来看，这种方法可以用于研究广泛的运动蛋白，其中许多与人类疾病密切相关，如运动神经元退行性疾病。

 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.