The long range goals are to determine the amino acid sequence of those peptides required for protein synthesis initiation and elongation in eukaryotes and attempt to relate amino acid sequence to the structure and function of each peptide. There is little information about the chemical characterization of the protein factors required for protein synthesis due to the scarcity of these proteins and the difficulty in acquiring sequence information with small amounts of material. As a result of recent modifications in purification procedures, it has become possible to have 10 to 100 mg quantities of several factors available (EF-lAlpha, eIF-2, eIF-4A). This combined with modifications in established sequencing techniques, make it now possible to analyze peptides with 2 to 10 nanomoles of material without resorting to the use of the """"""""microsequenator"""""""". In addition, the ability to screen expression cDNA libraries has made it possible to isolate cDNA clones in the absence of any sequence information. From either methodology, amino acid sequence or antibody, cDNA clones can be obtained and the entire """"""""native"""""""" amino acid sequence determined. Subsequently, limited amino acid sequencing can be used to determine the precise sequence of the amino and carboxyl terminii. Subsequent studies will attempt to identify specific functional regions of the protein which interact with ATP, GTP, mRNA, amino acyl-tRNA, other factors, or the ribosome. These regions will be identified using a variety of radioactive affinity labels of ATP, GTP, mRNA or aminoacyl-tRNA and by """"""""footprinting"""""""" for the analysis of the interaction of a given factor with other factors or ribosomal subunits. Having thus marked specific regions of the proteins, the proteins will be cleaved with amino acid specific proteases or chemical reagents followed by amino acid sequence analysis of purified radioactive peptides. The exact method of analysis of each factor will depend on the stability of the peptide-ligand bond and the availability of convenient cleavage points which would be known from the amino acid sequence of the unmodified protein.
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