Characterization of a Limited Trypsin Digestion Form of Eukaryotic Elongation

The involvement of the first 69 amino acids of eukaryotic elongation factor l a (EF-la) from rabbit reticulocyte in GTP and aminoacyl-tRNA binding has been analyzed by a variety of techniques. EF-la was subjected to limited trypsin digestion, which cleaved predominantly at residues 36 and 69. A digested form of Eecherichia coli EF-Tu, similar to the one used for this study, has been characterized by x-ray crystallography and is used as a structural model for EFla. This form of EF-la bound E. coli Phe-tRNAPhe similar to the wild type protein, but lacked activity in phenylalanine polymerization with poly(U)-programmed ribosomes. These results were obtained regardless of whether or not loosely associated N-terminal peptides were removed by gel filtration chromatography. The digested EF-la also shows reduced GTPactivity, but the activity is stimulated by both ribosomes and aminoacyl-tRNA. Binding of EF-la to the 80 S ribosome, as determined by association of reductively methylated protein through Sepharose 6B chromatography, is reduced approximately 7-fold for the limited digested form of the protein. Limited digested EF-la can, however, be photo-cross-linked with GTP and 3'p-azido-GTP similar to intact EF-la. Chemical crosslinking with oxidized GTP, fluorosulfonylbenzoylGTP, or with tram-diaminedichloroplatinum(I1) and GPT, shows a similar modification of both intact and limited digested EF-la. In order to further localize the modification site with the GTP reagents and assure that modification was not occurring in the first 69 amino acids, intact EF-la was modified with these same reagents. Limited trypsin digestion of modified protein indicates that none of these reagents crosslinks GTP to the first 69 amino acids of EF-la, which includes the first GTP binding consensus element, GXXXXGK.