On the structural specificity of puromycin binding to Escherichia coli ribosomes.
We have examined the structural specificity of the puromycin binding sites on the Escherichia coli ribosome that we have previously identified [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry 19, 3809-3817, and references cited therein] by examining the interactions of a series of adenine-containing compounds with these sites. We have used as measures of such interactions 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 two of these measures we have made extensive use of a recently developed high-performance liquid chromatography (HPLC) method for ribosomal protein separation [Kerlavage, A. R., Weitzmann, C., Hasan, T., & Cooperman, B.S. (1983) J. Chromatogr. 266, 225-237]. We find that puromycin aminonucleoside (PANS) contains all of the structural elements necessary for specific binding to the three 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. Our current results reinforce our previous conclusion that photoincorporation into L23 takes place from the A' site within the peptidyl transferase center and lead us to speculate that the S14 site might be specific for the binding of modified nucleosides. They also force the conclusion that puromycin photoincorporation proceeds through its adenosyl moiety.
D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism*; D010777:Photochemistry; D011691:Puromycin / Q000378:metabolism*; D011692:Puromycin Aminonucleoside / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D012269:Ribosomal Proteins; D011691:Puromycin; D011692:Puromycin Aminonucleoside