Characterization of a limited trypsin digestion form of eukaryotic elongation factor 1 alpha.
The involvement of the first 69 amino acids of eukaryotic elongation factor 1 alpha (EF-1 alpha) from rabbit reticulocyte in GTP and aminoacyl-tRNA binding has been analyzed by a variety of techniques. EF-1 alpha was subjected to limited trypsin digestion, which cleaved predominantly at residues 36 and 69. A digested form of Escherichia 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 EF-1 alpha. This form of EF-1 alpha 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-1 alpha also shows reduced GTPase activity, but the activity is stimulated by both ribosomes and aminoacyl-tRNA. Binding of EF-1 alpha 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-1 alpha can, however, be photo-cross-linked with GTP and 3'-p-azido-GTP similar to intact EF-1 alpha. Chemical cross-linking with oxidized GTP, fluorosulfonylbenzoyl-GTP, or with trans-diaminedichloroplatinum(II) and GPT, shows a similar modification of both intact and limited digested EF-1 alpha. 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-1 alpha was modified with these same reagents. Limited trypsin digestion of modified protein indicates that none of these reagents cross-links GTP to the first 69 amino acids of EF-1 alpha, which includes the first GTP binding consensus element, GXXXXGK.
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Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
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D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D008969:Molecular Sequence Data; D020648:Peptide Elongation Factor 1; D010444:Peptide Elongation Factor Tu / Q000737:chemistry; D010445:Peptide Elongation Factors / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D014176:Protein Biosynthesis; D012360:RNA, Transfer, Phe / Q000378:metabolism; D011817:Rabbits; D012270:Ribosomes / Q000378:metabolism; D013329:Structure-Activity Relationship; D014357:Trypsin
D000345:Affinity Labels; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D010446:Peptide Fragments; D012360:RNA, Transfer, Phe; D014357:Trypsin; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu