Affinity labeling of a reactive sulfhydryl residue at the peptidyl transferase P site in Drosophila ribosomes

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Abstract
An affinity label was prepared that is specific for the P site of an eukaryotic peptidyl transferase, that of D. melanogaster. It has the sequence C-A-C-C-A-(Ac[3H]Leu) with an Hg atom added at the C-5 position of all 3 cytosine residues (referred to as the mercurated fragment). This label is an analog 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 an Hg-S bond, as judged by its lability in the presence of thiol reducing agents. The major ribosomal protein labeled at the P site of D. malanogaster was 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 eukaryotes. Conservation of some of the overall properties of the P site proteins from these organisms is suggested.

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