The initiation of translation in eukaryotes is an extraordinarily complex process involving the action of at least 26 non-ribosomal polypeptides and requiring energy input from the hydrolysis of both ATP and GTP. At the end of the initiation process, a ribosome is assembled at the correct initiation codon of an mRNA with a methionyl initiator tRNA bound in its peptidyl (P) site and the second aminoacyl tRNA bound in its acceptor (A) site. At this stage the ribosome is active and is poised to catalyze the formation of the first peptide bond. While many of the steps in the initiation process have been delineated and the roles of some of the initiation factors established, little is known about the molecular mechanics of this fundamental biological process: how do the interactions among and motions of the components of the translational machinery catalyze and coordinate each of the steps required to build an initiation complex? This proposal outlines physical biochemical approaches for dissecting the pathway of eukaryotic translation initiation in vitro. These approaches will be used to establish a kinetic and thermodynamic framework describing each step in the initiation process. Such a framework will be crucial for attempts to understand the molecular mechanics of translation initiation. These techniques and the kinetic and thermodynamic framework will be used to probe the molecular roles of structural features of the mRNA that are known to be important for the initiation of translation: the 5'-7-methylguanosine cap structure, the 3'-poly(A) tail, structure in the 5'-untranslated region and the sequence surrounding the initiation codon. Experiments are also described to explore the structural and biochemical bases for a potential new step in the initiation process, a GTP-dependent activation of the 80S ribosomal complex. The studies outlined in this proposal will lay the foundation for many years of work aimed at understanding the molecular basis for each event required for eukaryotic translation initiation.