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



 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.