Affinity labeling of a reactive sulfhydryl residue at the peptidyl transferase P site in Drosophila ribosomes.
An affinity label has been prepared that is specific for the P site of a eucaryotic peptidyl transferase, that of Drosophila melanogaster. It has the sequence C-A-C-C-A-(Ac[3H]Leu) with a mercury atom added at the C-5 position of all three cytosine residues (referred to as the mercurated fragment). This label is an analogue of the 3' terminus of N-acetylleucyl-tRNA. The mercurated fragment binds specifically to the P site of peptidyl transferase. It participates fully in peptide bond formation as judged by its ability to transfer N-acetylleucine to puromycin with at least the same efficiency as a nonmercurated fragment. Once bound to the P site, the mercurated fragment reacts covalently with a ribosomal protein(s). This affinity-labeling process can be effectively competed by nonmercurated fragment, which indicates a site-specific reaction. The covalent attachment of the affinity label to a ribosomal protein(s) occurs through the formation of a mercury-sulfur bond, as judged by its lability in the presence of thiol reducing agents. The major ribosomal protein labeled at the P site of D. melanogaster was found to be a small, basic protein. The electrophoretic behavior of this protein parallels that of major P site proteins found in Escherichia coli ribosomes and in other eucaryotes. These results suggest conservation of some of the overall properties of the P site proteins from these organisms.
D000217:Acyltransferases / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D004331:Drosophila melanogaster / Q000201:enzymology*; D007814:Larva / Q000201:enzymology; D007930:Leucine / Q000032:analysis; D008628:Mercury; D010458:Peptidyl Transferases / Q000378:metabolism*; D011485:Protein Binding; D012343:RNA, Transfer; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000032:analysis; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000345:Affinity Labels; D012269:Ribosomal Proteins; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D012343:RNA, Transfer; D000217:Acyltransferases; D010458:Peptidyl Transferases; D008628:Mercury; D007930:Leucine