In eukaryotes, assembly of a ribosome on an mRNA (translation initiation) requires the action of at least 26 non-ribosomal polypeptides and energy input from the hydrolysis of both ATP and GTP. We are trying to understand the molecular mechanics of this extraordinarily complex process. To do this, we are using techniques from biophysics and mechanistic enzymology and collaborate closely with yeast geneticists studying the same problems. Current projects include: 1. Kinetic and thermodynamic analysis of the steps in translation initiation in a reconstituted yeast translation system using a wide variety of biophysical techniques (e.g., fluorescence spectroscopy); 2. Enzymology of isolated initiation factors; 3. Investigations of the mechanistic roles of structural elements in the mRNA and initiator tRNA such as the 5'-7-methylguanosine cap, 3'-poly(A) tail and the consensus sequence around the initiation codon in the mRNA, and conserved bases and modifications in the initiator tRNA. We hope that dissection of the initiation process into individual steps and measurement of the physical parameters that govern each of these steps will lead towards an understanding of the molecular interactions and events responsible for the assembly of the eukaryotic translational machinery.