This project focuses on the role of the ribosome as a molecular motor. During protein synthesis the ribosome moves towards the 3'-end along messenger RNA (mRNA), one codon at a time, driven by the hydrolysis of GTP. It reads the genetic information encoded by mRNA, recruits the appropriate transfer RNA and associated aminoacid, and pieces together a protein, thus linking genotype and phenotype. It is not well understood how the ribosome, a complex structure consisting of over 50 proteins and several ribosomal RNAs, works as a molecular motor and moves in coordinated fashion to maintain the reading frame. To answer such questions we will develop an in vitro motility assay and record translocation of individual ribosomes at nanometer-scale resolution. Ribosomes will be attached to a microscope coverslip and the displacement of a single ribosome will be derived from the movement of a microscopic bead, which has been attached to the mRNA molecule. Bead velocity will be directly related to ribosome velocity and translation rate. A special microscope equipped with optical tweezers and nanometer-sensitive position sensors will be constructed to measure the forces produced by individual ribosomes and to investigate how ribosome velocity is affected by mechanical load. Such information is essential for constructing physical or mathematical models of ribosome movement. The proposed research opens the way towards a variety of experiments at the single molecule scale, which are specifically aimed at protein synthesis and ribosome mechanics or dynamics. Such experiments will shed light on how this naturally occurring protein-making machine works, and might provide valuable information for designing similar man-made nanomachines.
