Study of Helicase Activity of the Ribosome using Single-Molecule FRET

Abstract

Secondary and tertiary structures of mRNA may become barriers to the ribosome during translation. Previous studies have shown that the ribosome itself is capable of opening the base-paring of mRNA. More recently, by using optical tweezers, Qu et al. have shown that this unwinding process involves two kinds of active mechanisms, in which the ribosome destabilizes and mechanically unwinds the encountered junctions on mRNA. However, detailed interaction between the ribosome and mRNA at the junction remains to be elucidated. In this study, we aim to utilize TIRF (total internal reflection fluorescence) microscopy to detect FRET signals at single-molecule level, so that we can observe the stepwise unwinding process during translocation in real-time. We have purified all the translation factors as well as the ribosome required for this system from Escherichia coli. We have also constructed a template to mimic RNA duplex structures by pairing a Cy5-labeled DNA oligonucleotide to a Cy3-labeled mRNA. Then, the duplex will be mixed with the defined in vitro translation system that allows us to control each translocation step of the ribosome by adding one unique aminoacyl-tRNA at a time. We expect the FRET efficiency of the dye pair will fluctuate as the ribosome translocates through the RNA-DNA junctions.