Photoinduced affinity labeling of the Escherichia coli ribosome puromycin site

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Abstract
The photoincorporation of puromycin into E. coli ribosomes was studied in detail. Incorporation into protein L23 as a function of puromycin concentration follows a simple saturation curve and is specifically blocked by structural and functional analogs of puromycin, thus demonstrating that such incorporation proceeds via an affinity labeling process. Incorporation into L23 becomes more specific as the light fluence is reduced, indicating that such incorporation takes place from a native rather than light-denatured puromycin site. L23 remains the major labeled protein using ribosomes prepared by several procedures, suggesting the conservative nature of the site. Evidence is presented for affinity labeling of S14 and of a site in the RNA fraction of the 50S particle. Specific incorporation appears to proceed with an anomalously high quantum yield. The detailed photochemical mechanism is not understood, although 8-alkylation of the purine moiety was excluded. Incorporation is largely inhibited in the presence of thiol reagents.