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J Am Chem Soc. 2017 Jan 11;139(1):262-268. doi: 10.1021/jacs.6b10387. Epub 2016 Dec 29.

RNA Scanning of a Molecular Machine with a Built-in Ruler.

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Department of Biophysics, Johns Hopkins University , Baltimore, Maryland 21218, United States.
Physics Frontier Center (Center for Physics of Living Cells), University of Illinois , Urbana, Illinois 61801, United States.


Advanced single-molecule techniques have enabled tracking of nanometer-scale movements of DNA and RNA motor proteins in real time. Previously, we reported an ATP-independent diffusion of transactivation response RNA binding protein (TRBP) on dsRNA, yet the mechanistic details remain elusive. Using single-molecule fluorescence assays, we demonstrate that the diffusion activity of TRBP is coordinated by an independent movement of two subdomains, dsRBD1 and dsRBD2, in which the diffusion distance is determined by the length of a flexible linker domain that connects the two dsRBDs. When the linker is shortened, the diffusion distance is reduced proportionally, suggesting a ruler-like function of the linker domain. Diffusion stalls upon encountering a physical barrier in the form of an RNA:DNA hybrid segment or bulky secondary structures, indicating a dsRNA scanning mode of TRBP. The results display a plausible mechanism of TRBP in scanning for pre-miRNA or pre-siRNA as proper substrates for the RNAi pathway.

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