The Molecular Mechanics of Start Site Recognition in Eukaryotic Translation

Abstract

This talk will describe recent experiments aimed at understanding the molecular mechanisms underlying the recognition of the translation initiation site in an mRNA by the eukaryotic protein synthesis machinery. This is arguably the most important reading of the genetic code during gene expression because if it goes awry a miscoded protein will be produced. Upon recognition of the initiation codon, a signal is sent to the central G protein initiation factor eIF2 to irreversibly hydrolyze its bound GTP and release the methionyl initiator tRNA into the P site of the small ribosomal subunit. This event is the first committed step in translation initiation; after it happens the complex must proceed with initiation at that point on the mRNA or abort the process. Thus the irreversible hydrolysis of GTP by eIF2 must be regulated exquisitely carefully such that it does not happen at the wrong place on the mRNA but happens very rapidly at the right place. We have been exploring the molecular mechanics of the formation of the 43S·mRNA pre-initiation complex, its identification of the start codon in the mRNA, and the triggering of irreversible GTP hydrolysis by eIF2. The work employs a reconstituted yeast translation initiation system, and the roles and mechanisms of each of the key components of the initiation machinery required for these central steps are being elucidated through a thermodynamic and kinetic dissection of the pathway. In collaboration with Alan Hinnebusch's lab at the NIH, we have also been studying a number of mutant versions of initiation factors that produce well characterized phenotypes in vivo, such as reduction in the fidelity of initiation codon recognition. These studies thus synergistically harness the power of yeast genetics and molecular biology to the detailed biophysical and biochemical studies possible in vitro.