Browning 9406601 The specific objectives of this proposal are to: 1) Determine the functional domains of the subunits of wheat germ eIF-(iso)4F and EIF-4F. The subunits of eIF-(iso)4F have been expressed in E. coli and recombined to form an enzymatically active complex. Mutations will be made in the subunits of eIF-(iso)4F and eIF-4F and tested in an in vitro wheat germ translation system and several in vitro assays that measure partial reactions, i.e., ATP hydrolysis, RNA unwinding, cross-linking to the mG cap and binding of mRNA to 40S ribosomal subunits. 2) Complete the partial cDNA sequence of wheat eIF-4B and express this protein in E. coli. The missing portion (~100-200 nucleotides) of the cDNA for eIF-4B will be obtained by RACE-PCR, inverse PCR or genomic sequencing. The cDNA will be expressed in E. coli and the enzymatic activity determined in the assays described above (Specific Objective 1). 3) Complete the cDNA sequence for the subunits of Arabidopsis eIF-4F, p28 of eIF- (iso)4F and eIF-4B and express these proteins in E. coli. We will continue to screen using antibodies to wheat factors and wheat cDNA sequences. Different regions of the wheat cDNAs will be used to prepare probes for screening. The cDNAs for Arabidopsis factors will be expressed in E coli. The activity of the expressed Arabidopsis factors will be measured in the assays described above. The completion of this goal will set the stage for future work in vivo exploring the control of expression of the inhibition factors and regulation of protein synthesis in plants. %%% The long term goal of this project is to elucidate the steps in the initiation of protein synthesis in plants. Specifically we want to determine the structural aspects of plant protein synthesis initiation factors (eIFs) that are important for the interaction of the factors with each other, with mRNA and ATP, and for the binding of mRNA to 40S ribosomal subunits. The project goals are to: 1) Determine the fun ctional domains of the subunits of eIF-(iso)4F and eIF-4F using mutagenesis. 2) Complete the cDNA sequence of wheat eIF-4B and p220 of wheat eIF-4F and express these proteins in E. coli. 3) Complete the cDNA sequence for the subunits of Arabidopsis eIF-(iso)4F and eIF-4F and eIF-4B and express these proteins in E. coli. The results of this research will enhance our understanding of these important proteins and provide valuable information about their structure and function. ***
