Structure of Plant Protein Synthesis Initiation Factors

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The project goals of this research proposal are: 1) to clone and sequence the cDNAs for the two subunits of wheat germ eukaryotic initiation factor (eIF)-4F (p220 and p26) and the two subunits of its isozyme form, eIF-(iso)4F (p82 and p28). 2) to identify the m7G cap binding site of p26 and p28 by comparing the sequences of these polypeptides to each other an to m7G cap binding proteins from other cells (yeast, mouse, human). In addition, a comparison of the sequences of p220 and p82 will be made in an effort to identify the ATP binding site, mRNA binding and RNA helicase domains of these peptides. 3) to clone an sequence the cDNAs for eIF-4A and eIF-4B, to compare these sequences to those of obtained from other eukaryotic cells, and to attempt to identify functional domains. 4) to use the wheat cDNA clones to determine the number, location and sequence of the genes in Arabidopsis for eIF-4A, eIF-4B and the subunits of eIF-4F and eIF-iso)4F. 5) to use the wheat cDNA clones for eIF-4A, eIF-4B and the subunits of eIF-4F and eIF-(iso)4F to determine the level of expression of these genes during various stages of plant development. To achieve these goals, various methods to screen a wheat cDNA expression library will be used. Antibodies to the initiation factors are available, as well as some knowledge of peptide sequences which will be used to design oligonucleotide probes. DNA sequencing, Southern and Northern blot analysis and quantitation of mRNA will be carried out by established techniques. It is not known what structural features of eIF-4A, eIF-4B and eIF-4F are important for their function. Completion of these goals will provide valuable information with regard to the structural features of these polypeptides that are important for the recognition of the 5' m7G cap of mRNA, for binding to mRNA, for ATP-dependent RNA helicase activity and for binding of mRNA to ribosomes.