Structure and Function of Plant Protein Synthesis Initiation Factors

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The initiation of protein synthesis in all eukaryotes is an intricate process requiring at least 8-10 known initiation factors. The long term goal of the PI's laboratory is to elucidate a detailed molecular description of this process in plants. Towards this end they have isolated and cloned plant initiation factors to determine their biochemical properties and structures, and ultimately to map their physical and functional interactions with each other, mRNA and ribosomes. The overall objective of this research is to obtain an understanding of the function of two forms of eIF4F in plant translation initiation. Three specific questions will be explored. First, how does the molecular composition of eIF4F and eIF(iso)4F complexes affect specificity of mRNA translation, and what features of a mRNA (5' UTR, 3' UTR or coding region) affect discrimination by eIF4F and eIF(iso)4F? Plants have a second novel form of eIF4F, eIF(iso)4F. Some mRNAs are translated poorly in vitro by eIF(iso)4F, whereas others do not show a preference for eIF4F or eIF(iso)4F. The subunit(s) of eIF4F and eIF(iso)4F that confer specificity of mRNA translation will be determined using mixed subunit complexes (eIF4G/eIF(iso)4E and eIF(iso)4G/eIF4E). In addition, the portions of the mRNA (5' UTR, 3' UTR and/or coding region) that are necessary for discrimination by eIF4F and eIF(iso)4F will be determined using chimeric mRNAs. Second, is eIF(iso)4F redundant or necessary for plant survival? Genetic methods will be used to determine whether knockouts or gene silenced eIF(iso)4F subunits, eIF(iso)4E, and eIF(iso)4G produce any obvious phenotypes and/or are necessary for viable plants. Finally, how does the binding of eIF4G affect eIF4E structure and affinity for mRNA? A peptide derived from eIF4G that contains the eIF4E binding site will be used to make a complex to study the changes in eIF4E structure upon its binding. Further studies will be carried out to measure changes in mRNA affinity for the complex compared to eIF4E.Protein synthesis is an essential molecular process for all cells. An understanding of the interactions of the factors, ribosomes and mRNA is necessary to integrate this process and its regulation with other cellular processes. It is clear that some aspects of the initiation of protein synthesis are very similar among all higher organisms, but that there are some fundamental differences. The presence of the isozyme form of eIF4F suggests that plants may have retained a more ancient form of the eIF4F complex or evolved a second complex for some specific purpose or function in plants. A clear understanding of how plants manufacture their proteins will help in the development of plants with higher nutritive value or as living factories for the production of protein pharmaceuticals.This project is supported by the Biochemistry of Gene Expression Program and the Molecular Biochemistry Program in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.