The formation of polyadenylated 3' termini is an essential step in the biogenesis of eukaryotic mRNA. In mammals, this process has been examined extensively in vivo, as well as in cell-free systems, and has identified specific sequences for polyadenylation. in lower eukaryotes such as yeast, less than 50% of the mRNAs examined to-date have an identifiable polyadenylation signal equivalent to the mammalian sequence. We believe, therefore that the formation of mRNA 3' ends in fungi may proceed by a mechanism significantly different than that which occurs in mammalian cells. This processing step could represent a novel target for the development of antifungal drugs. In Phase I we will construct 3 reporter plasmids to confirm that yeast polyadenylation signals differ from that of mammals. Deletion mutagenesis and an in vitro polyadenylation system will be used to define the yeast polyadenylation signal sequences. As part of the Phase II we will develop an in vivo system with the reporter plasmids to screen chemicals for inhibition of 3' end formation in yeast. Also in Phase II the in vitro system will be used to clone enzymes involved in 3' end formation and confirm that chemicals inhibiting in vivo systems actually inhibit 3' end formation.
