On the structural specificity of puromycin binding to Escherichia coli ribosomes

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Abstract
The structural specificity of the puromycin binding sites on the Escherichia coli ribosome were examined by studying the interactions of a series of adenine-containing compounds with these sites. As measures of such interactions used were the inhibition of [3H]puromycin photoincorporation into ribosomal proteins from these sites, the site-specific photoincorporation of the 3H-labeled compounds themselves, and the inhibition of peptidyl transferase activity. For the first 2 of these measures extensive use was made of a recently developed high-performance liquid chromatography (HPLC) method for ribosomal protein separation. Puromycin aminonucleoside (PANS) contains all of the structural elements necessary for specific binding to the 3 major puromycin binding sites, those of higher affinity leading to photoincorporation into L23 and S14 and that of lower affinity leading to photoincorporation into S7. Although tight binding to the L23 and S7 sites requires both the N6,N6-dimethyl and 3''-amino groups within PANS, only the N6,N6-dimethyl group and not the 3''-amino group is required for binding to the S14 site. Photoincorporation into L23 takes place from the A'' site within the peptidyl transferase center and the S14 site might be specific for the binding of modified nucleosides. Puromycin photoincorporation proceeds through its adenosyl moiety.