The photoincorporation of puromycin into Escherichia coli ribosomes has been 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 analogues 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. In addition 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 purine moiety has been excluded. Incorporation is largely inhibited in the presence of thiol reagents.

ABSTRACT[3HJPuromycin and N4ethyl-2-diazomalonylX3H]puromycin are incorporated into E. coli ribosomes on irradiation at 253.7 nm. Both compounds incorporate into both protein and nucleic acid. Two-dimensional gel electrophoresis of ribosomal protein shows that L23 is the major protein labeled by puromycin. Although incorporation is clearly a complex process, evidence is presented that L23 is labeled via an affinity labeling process, thus placing L23 at the aminoacyl-tRNA receptor (A) site. N.(ethyl-2-diazomalonyl)puromycin is a ribosomal ligand, as shown by its inhibition of two ribosomal assays, but it is not a good puromycin analog, and it is unclear whether its incorporation, which proceeds via both carbene-dependent and carbene-independent processes, results from affinity labeling.Affinity labeling (1) has found wide application as a tool for mapping complex biological structures, and has recently been applied to ribosomes (2-15, 40, 41). This technique uses either electrophilic or photolabile derivatives of biological ligands to form covalent bonds with the appropriate biological receptors. The advantages, at least in principle, of photolabile derivatives have been discussed elsewhere (16). As part of a large-scale effort to use photolabile antibiotic derivatives to map ribosome function, we have synthesized N-(ethyl-2-diazomalonyl)puromycin and studied its covalent incorporation into ribosomes on photolysis. During the course of this work we found that puromycin itself will photoincorporate into ribosomes. This paper reports the results of initial studies on these two incorporation processes. MATERIALS AND METHODSPuromycin dihydrochloride was obtained from Sigma.[3H]Puromycin, labeled in the methoxyl protons, was obtained from New England Nuclear. Ethyl-2-diazomalonyl chloride was prepared as described previously (17). Ribosomes used in all incorporation experiments were prepared from Escherichia coli Q13 bacteria harvested in late log phase by the method of Kurland (18). N-(ethyl-2-diazomalonyl)puromycin (N-EDMpuromycin) was N-EDMpuromycin was characterized by its ultraviolet spectrum, which is the sum of two isolated chromophores, puromycin and ethyl-2-diazomalonamide (Xma. 260 nm, Emax 23,000 M-1 cm'1). Brief photolysis of N-EDMpuromycin at 253.7 nm results in the appearance of an ultraviolet spectrum identical to that of puromycin. Further proof that the amino group is the site of derivatization rather than either of the two hydroxyl groups comes from the observations that N-EDMpuromycin is stable toward release of ethyl-2-diazomalonic acid on treatment with 0.1 N NaOH, and that on prolonged standing in alkaline aqueous or alcoholic solutions, N-EDMpuromycin isomerizes into a compound which is not readily photolyzed and whose ultraviolet spectrum indicates that it is a 1,2,3 triazole (Xm. 272, em. 28,000) (17).Although the triazole cannot be readily converted to an ethyl-2-diazomalonamide in water, the reconversion proceeds smoothly on dissolving the triazole in a 6% acetic acid/ chloroform solution....

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