Characterization of Arabidopsis thaliana translation initiation factor eIF4F

Eukaryotic translation initiation is a complex process involving many factors. Two vital components of this system are eukaryotic initiation factors (eIF) 4E and 4G, which together form the complex eIF4F. eIF4E is the cap-binding subunit, interacting with the 7-methyl guanosine at the 5'-end of messenger RNA. There are multiple forms of this gene in Arabidopsis thaliana: eIF4E1, eIF4E2, eIF4E3, eIFiso4E, and novel cap-binding protein. The scaffolding component of the eIF4F cap-binding complex is eIF4G. This protein binds not only to eIF4E, but also to eIF3, eIF4A, and poly(A)binding protein. Three versions are found in Arabidopsis: eIF4G, eIFiso4G1, and eIFiso4G2. Plants are unique in that they are the only organisms to contain the isoform versions of these genes. A goal of the research described in this thesis is to determine the mechanisms by which plant eIF4F isoforms specifically select mRNAs for translation. This thesis describes the cloning of dicistronic Arabidopsis eIF4G with some members of the eIF4E cap-binding family. Comparison of the biochemical activities of these complexes with various mRNAs will generate more information about their specific functions. Analysis of Arabidopsis T-DNA insertion mutants is included to elucidate the role(s) of eIF4F, eIFiso4F, and their subunits in plant translation. The phenotype(s) of a mutant may provide clues as to the function of the protein(s) not expressed. The importance of the eIF4F complex to translation initiation makes it a target for regulation. These regulatory mechanisms include inhibition by cleavage, competitive inhibitors, phosphorylation, binding of partner proteins, and exploitation by viruses. Our hypothesis is that the eIF4E and eIF4G genes are also regulated at the level or translation. Part of this thesis describes preparation and sequencing of cDNAs for eIF4G, eIFiso4G1, eIFiso4G2, eIF4E1, eIF4E2, eIF4E3, eIFiso4E, and nCBP to identify full length 5'UTRs and determine if regulatory elements are present.Biochemistr