The objective of my research is to determine the mechanisms that control the initiation of protein synthesis in yeast. The goal of this proposal is to describe in genetic and molecular terms sequence and structural complexites in the HIS4 initiator region that govern translational initiation and specific interactions with the components of the translational initiation complex. The following specific aims are directed at achieving this goal: 1) The HIS4 translational initiation region will be systematically mutated emphasizing simple base changes in the non-coding sequences and insertion of secondary structure that might affect ribosomal recognition of the mRNA, scanning of the leader and ribosomal selection of the AUG start condon. 2) These translationally defective mutants will then be used in reversion tests which restore the ability to express HIS4 using an effective selection scheme to identify external suppressors. These external suppressors should define components of the initiation complex that have altered specificities for the mutant HIS4 region and therefore represent gene products that interact with the mRNA in a specific manner. 3) These external suppressors will be rigorously characterized genetically and in conjunction with the previously isolated sui1, sui2, and SUI3 external suppressors of initiator condon mutations at HIS4 and the cloned tRNA-1-MET gene will be characterized at the molecular and biochemical level to determine the mechanism of suppression and the function of the suppressor gene products. Furthermore, the genetic identification of the components of the translational initiation complex will enable us to construct probes and devise genetic selection schemes that will allow a comprehensive analysis of the expression and regulation of their essential gene products. This work should be directly related to many studies in higher eukaryotes contributing valuable information into the basic control mechanisms that determine proper and efficient gene expression and may provide important insight into mechanisms that coordinate hormal cellular growth in yeast.
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