Affinity labeling of the P site of Drosophila ribosomes: a comparison of results from (bromoacetyl)phenylalanyl-tRNA and mercurated fragment affinity reactions.
The binding site of the peptidyl group of peptidyl-tRNA in the P site of Drosophila ribosomes was probed with (bromoacetyl)phenylalanyl-tRNA (BrAcPhe-tRNA). This affinity label binds specifically to the P site by virtue of its ability to participate in peptide bond formation with puromycin following its attachment to ribosomes. As many as nine ribosomal proteins may be labeled under these conditions; however, the majority of the labeling is associated with three large-subunit proteins and two small-subunit proteins. Two of the large-subunit proteins, L4 and L27, are electrophoretically very similar to the proteins labeled by the same reagent in Escherichia coli ribosomes L2 and L27. Reexamination by a different two-dimensional gel system of the ribosomal components labeled by a second P site reagent, the 3' pentanucleotide fragment of N-acetylleucyl-tRNA which is derivatized to contain mercury atoms at the C-5 position of all three cytosine residues, shows two major and three minor labeled proteins. These proteins, L10/L11, L26, S1/S4, S13, and S20, are likely present in the binding site of the 3' end of peptidyl-tRNA, a site that appears to span both subunits. These results have allowed us to construct a model for the protein positions in and near the peptidyl-tRNA binding site of Drosophila ribosomes.
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Molecular Biology Section, University of Southern California, Los Angeles 90089.
Pellegrini
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D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D004330:Drosophila / Q000235:genetics / Q000378:metabolism*; D008628:Mercury / Q000494:pharmacology*; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure
D000345:Affinity Labels; D012346:RNA, Transfer, Amino Acyl; D012269:Ribosomal Proteins; C032617:tRNA, N-bromoacetylphenylalanine-; D008628:Mercury