Function of Plant Protein Synthesis Initiation Factors: eIF4G and eIFiso4G

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Intellectual Merit: The aim of the project is to understand the mechanism by which plant cellsstart the process of making proteins. The model plant, Arabidopsis, provides excellent genetic tools to dissect the role of various cellular components in this process during normal plant growth and development as well as under a variety of environmental stresses such as changes of temperature, drought, or infection by plant pathogens. Although the mechanism of protein synthesis in plants is similar to that of animals or insects, some aspects are unique to plants. Many of these differences may be related to the fact that plants must interact with the environment while remaining in a single place and must obtain their energy through photosynthesis. A variety of genetic and biochemical tools will be applied to determine the specialized nature of how plant cells synthesize proteins. Biochemical and phenotypic analysis of T-DNA mutants of initiation factors eIF4G, eIFiso4G1 and eIFiso4G2 will be carried out, as well as analysis using tagged proteins (regulated by both native and CaMV 35S promoters) in various plant backgrounds. Environmental stressors (light, heat, cold, hormones, redox, etc.) will be applied to determine if any of the mutant or transgenic plants display phenotypes associated with particular stresses. Changes in protein expression will be assessed in the various mutants and transgenic expression lines and any proteins with altered expression will be identified. Several methods will be utilized to further identify any mRNA targets that are reduced on polysomes in the T-DNA knockout mutants for eIFiso4G1, eIFiso4G2 or eIF4G or that associate directly with these proteins or complexes. Those mRNAs identified from DNA array analysis of polysomes from mutant plant lines will be validated using using in vitro translation assays dependent upon the presence of eIFiso4F and confirmation by qRT-PCR of reduced polysome loading of the mRNAs in the mutant plant lines. Further validation will be done using in vivo expression of tagged versions of the identified mRNA?s coding region in plants lacking one or more of the genes for the subunits eIFiso4F. The mRNAs will also be used as ?bait? to pull down and identify additional RNA binding proteins.Broader Impact. High school, community college, undergraduate, graduate and post-doctoral students will be involved in this research project. Undergraduates will enter through the Freshman Research Initiative (FRI), a unique program at the University of Texas at Austin for training students in science, technology, engineering and math (STEM) disciplines. Beginning students will be taught the necessary skills to engage productively in research and will learning science by participation. The students in the FRI will be responsible for construction of in vitro expression clones for the mRNAs, construction of plasmids for over-expression of proteins and tagged proteins in plants and will be involved in the phenotype analysis of mutant plant lines. In addition to training future research scientists, this project will lead to new information about how plant cells make proteins. This knowledge may be applied to improve plants to better resist environmental insults or virus infection and to provide better quality protein for food for humans and livestock.