Single-molecule Fluorescence Microscopy Study Of The Ribosome Translation Process

Abstract

The ribosome is the molecular motor responsible for the protein synthesis within all cells. Ribosome motions along the messenger RNA (mRNA) to read the genetic code are asynchronous and occur along multiple kinetic paths. Consequently, observation and manipulation at the single macromolecule level is desirable to unravel the complex dynamics involved. In this communication, we present the study of translation kinetics of single ribosomes via the direct observation of fluorescent amino-acid incorporations.In order to study the kinetics of amino-acid incorporation inside the growing protein by the ribosome, we use a home-made total internal reflection single-molecule fluorescence microscope (TIRFM). The mRNA-ribosome complex is attached to a polyethylene glycol modified glass coverslip surface by a streptavidin-biotin linkage. The ribosome is labelled with a quantum dot (QD) in order to localize it on the surface while a specific amino acid (lysine) is marked with Bodipy-FL [1]. This fluorescent dye is small enough to enter the ribosomal channel thus leaving intact ribosomal activity. The protein synthesis is observed in real time as the labelled amino acids are incorporated into the polypeptidic chain by the co-localization of QD and Bodipy-FL fluorescence signals.We will discuss the future application of this technique to single-molecule observation of the translation process, proof reading or even protein folding.Reference[1] K. Perronet, P. Bouyer, N. Westbrook, N. Soler, D. Fourmy, and S. Yoshizawa. Journal of Luminescence, 127, 264, 2007.