Kinetic Dissection of Fundamental Processes of Eukaryotic Translation Initiation in Vitro

Approaches have been developed for the kinetic dissection of eukaryotic translation initiation in vitro using rabbit reticulocyte ribosomes and a crude preparation of initiation factors. These new approaches have allowed the kinetics of formation of the 43S and 80S ribosomal complexes to be followed and have substantially improved the ability to follow formation of the first peptide bond. The results suggest the existence of a new step on the initiation pathway that appears to require at least one additional factor and the hydrolysis of GTP and may prepare the 80S complex for the formation of the first peptide bond. The initial kinetic framework and methods developed herein will allow the properties of individual species along the initiation pathway to be probed further and will facilitate dissection of the mechanistic roles of individual translation factors and their interplay with RNA structural elements.