Single-Molecule Fluorescence Observations of Eukaryotic Translation

Abstract

For the two last decades, structural and single-molecule investigations of the prokaryotic ribosome have provided a better understanding of the highly dynamic nature of translation. Eukaryotic ribosome has received far less attention, most of the conclusions being extrapolated from prokaryotic data. Although both ribosomes share functional and structural features, eukaryotic ribosome contains many additional domains of unknown function. Applying single-molecule techniques to eukaryote ribosomes would clarify the function of these additional domains.In order to study structural rearrangement of the ribosome during eukaryotic elongation, we bypass canonical initiation using an initiation factor free Internal Ribosome Entry Site (IRES) enabling elongation in the desired reading frame. Ribosomal subunits, purified from Rabbit Reticulocyte Lysate (RRL), are first bound to the IRES. Ribosome-mRNA complexes are then fixed on a microscope coverslip via the 5'end of the mRNA. All factors necessary for elongation are provided by injection of RRL. using single molecule Total Internal Reflection Fluorescence (TIRF) microscopy, we monitor elongation thanks to a fluorescently labelled oligonucleotide hybridized on the mRNA. The oligonucleotide is detached by the helicase activity of a translating ribosome. The evanescent wave generated by the TIRF apparatus allows solely the detection of molecules close to the surface. In this configuration, probe detachments induce the loss of fluorescence signals which are used to measure the distribution of translation speed. A thorough care is given to photobleaching during data analysis. Controls using antibiotics further show that the loss of fluorescence is a signature of a single ribosome activity.