Translation of leaderless mRNAs in the archaeon Sulfolobus solfataricus

Very little is currently known about the mechanism of translation in archaea. The translational apparatus of some characterized archaea is a mosaic of bacterial and eukaryal features in that translation factor homologues of both bacteria and eukarya are present in these organisms. Herein, we will explore features of the translation initiation factors, aIF1A, IF2 and aIF2 of Sulfolobus solfataricus. A major emphasis of this grant application is devoted to translation of archael leaderless mRNAs which start directly with the AUG-initiating codon. They appear to be prevalent in archaea and seem to be particularly abundant in the crenarchaeota S. solfataricus. This organism was chosen because its genome sequence is near to completion and accessible to the scientific community. This should allow cloning and purification of translation initiation factors. Moreover, vector systems for S. solfataricus are available which allows exploration of the functions of these initiation factors in their natural host. The aim of this work is twofold: First, we will attempt to set up an in vitro system with S. solfataricus ribosomes to assess the functions of the initiation factors to be purified during the course of this study in vitro. Second, we will attempt to over-express separately the bacterial and eukaryal homologues of the genes encoding initiation factor 2 in S. solfataricus. We will test whether the prokaryal or eukaryal homologue differentially affect gene expression in this organism. If so, the target genes will be identified using proteomics. It is anticipated that from an evolutionary perspective these studies will shed new light on the fundamentally important process of translation.