Single Molecule Study on RNA Chaperone Hfq and Development of Autofocusing System for Single Molecule Fluorescence Imaging

Abstract

Hfq is a bacterial RNA chaperone stimulating specific RNA-RNA interactions by inducing RNA unwinding or RNA-RNA base-paring. However the detailed molecular mechanism of how Hfq selectively achieves completely two opposite functions—unwinding, and base-paring—depending on RNA substrates is still unclear. Here, we developed single-molecule fluorescence assay monitoring Hfq-mediated RNA interactions. RNA fragments from DsrA small non coding RNA and rpoS mRNA of E. coli were used as a model system. Our real time observations reveal that Hfq stimulates DsrA:rpoS base-paring by simultaneously binding both RNAs using same RNA binding site on its proximal surface. The competition for the same binding site of the two RNAs makes the RNA-Hfq interaction dynamic, but drives efficient base-paring by properly aligning two RNAs in close proximity. When Hfq-binding sequence presents only in one of the two RNAs, RNA unwinding process dominates over the base-paring process. Single-molecule fluorescence imaging has greatly contributed to our understanding of many bio-molecular systems. While reactions occurring in the range of several minutes can be readily studied using conventional single-molecule fluorescence microscopes, data acquisition for longer time scales is hampered by the focal drift of high numerical aperture objectives, which should be corrected in real time. Here, we developed a robust autofocusing system based on optical astigmatism analysis of single-molecule images. Compared to the previously developed methods, our approach has a merit of simplicity in that neither fiducial makers nor an additional laser-detector system is required. As a demonstration, we observed B-Z transition dynamics occurring for several hours.