The end of the beginning: Eukaryotic ribosomal subunit joining and the combined roles of initiation factors 5B and 1A

The main goal of translation initiation is to position an initiator methionyl-tRNA base-paired to the AUG start codon in the peptidyl site of the ribosome, thus establishing the correct reading frame for translation and ensuring proper synthesis of the encoded polypeptide. This process is extremely complicated in eukaryotes, requiring at least 26 individual polypeptides as well as the energy of both ATP and GTP hydrolysis. Genetic and qualitative biochemical studies have already shed light on the roles of many of these components, but the molecular mechanics of the process have remained elusive. We therefore set out to elucidate the mechanisms by which the many components of translation initiation in eukaryotes find the site at which to begin translation and then assemble a translationally-competent ribosomal complex. Using an in vitro translation initiation system reconstituted from S. cerevisiae components, we have investigated the functional relevance of the eukaryotic-specific interaction between eukaryotic initiation factor (eIF) 1A and eIF5B, two factors that are universally conserved in translation initiation. eIF1A has been shown to participate in the early steps of translation initiation and was thought to dissociate from the initiating ribosome after start codon selection. In contrast, eIF5B, a GTPase, joins the initiating ribosome at a later step, facilitating coupling of the large ribosomal subunit to the complex. Using a combination of quantitative biochemical and biophysical techniques, we have shown that the eIF1A-eIF5B interaction accelerates ribosomal subunit joining by recruiting and positioning eIF5B on the initiating ribosome. In addition, eIF1A remains bound to the ribosome even after subunit joining and is removed only upon GTP hydrolysis by eIF5B, which alters the conformation of the initiation complex, and thus accelerates eIF1A release. Taken together with previous work from our lab and others, these data suggest a model of eukaryotic translation initiation in which eIF1A is present throughout initiation, serving as a molecular controller that coordinates the nature and timing of the myriad interactions required for initiation complex formation.