The long term goal of my research is to determine the mechanisms thatcontrol the initiation of protein synthesis in yeast The goal of thisproposal is to describe in genetic and molecular terms components of thetranslation initiation complex that functionin mediating ribosomalbinding of mRNA, scanning of the leader region and recognition of theinitiator region as well as the mechanisms by which these components maycontrol the initiation process. The following specific aims are directedat achieving these goals: 1 Genetic reversion analysis has identified ten different suppressorgenes which when mutated restore HIS4 expression despite a stem-looptranslational barrier in the leader region. These as well as othersuppressor genes, isolated for their ability to suppress stem-loopmutations at alternative positions in the leader, will be characterized todetermine if their gene products function at the time of ribosomal bindingor scanning of mRNA. 2 The functional significance of the 7methylGppp cap structure at the 5' end of mRNA for translation initiation in yeast will be investigated bygenetic methods. The HIS4 gene has been put under the control of the rDNApromoter as an attempt to produce "capless" HIS4 mRNA in yeast This rHIS4mRNA will be characterized for the absence of a posttranscriptionalmodification at the 5' end of the message and rHIS4 strains will be usedto identify genetic suppressors as potential candidates for gene productsthat might function in promoting ribosomal binding to mRNA. This geneticapproach may also provide a productive way to explore alternative in vivomechanisms for ribosomal binding to mRNA. 3 Characterization of the suil suppressor locus has shown that this geneproduct is functionally related to the sui2 and SUI3 suppressor locus butnot a subunit of elF-2. As an attempt to gain insight into the function ofsuil we will use molecular and biochemical approaches to try to establishwhat gene products suil might interact with or if it is part of a complexwith eIF-2 that is important forthe start site selection process. Inaddition, we will continue with our characterization of recessive lethal, SUI suppressor mutants that we isolated in diploid cells to determine ifother components of the initiation complex are essential for mediatingstart site selection. 4 The sui2 gene which encodes the alpha subunit of eIF-2 isphosphorylated at three other Ser residues by caesin kinase Il We willnow determine whether these phosphorylation events, or any other as yetuncharacterized phosphorylation events, are of any consequence to eIF-2function in yeast or of any physiologic significance as a mechanism ofcontrolling translation initiation. In light of similarities we have established among the yeast and humaninitiation processes and their translation factors, as well as a commontranslational control mechanism that is important for anti-viral responsein humans, our studies of the basic translation initiation process inyeast should continue to prove valuable to fundamental control processesin all eukaryotes that are of biomedical importance.
Control of translation initiation in yeast.
Genetic, molecular and biochemical analysis of translation initiation in yeast.
Casein Kinase, Fungal Genetics, Genetic Translation, Laboratory Rabbit, Mutant, Phosphorylation, Ribosome, Saccharomyces Cerevisiae, Suppressor Mutation, Translation Factor
