Photoincorporation of puromycin and N-(ethyl-2-diazomalonyl)puromycin into Escherichia coli ribosomes.

Abstract

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....

Photoincorporation of tetracycline into Escherichia coli ribosomes

“…ml-'. With the second lamps, samples were irradiated in vertical tubes as in [2] with an incident dose of 11 X 1016 quanta . s-l. ml-r. All photolyses were performed in standard TMK buffer (50 mM Tris, 10 mM MgCls or MgOAcz, loo-150 mM KC1 (pH 7.5-7.6)) at #'C.…”

“…It has been shown that native puromycin photoincorporates into specific sites on the E. coli ribosomes [2]. Taking note of the intrinsic photolability of tetracycline [3,4], we decided to investigate the possible photoincorporation of native [3H]TC into E. coli ribosomes.…”

Photoaffinity labeling of Escherichia coli ribosomes with an aryl azide analogue of puromycin

“…The puromycin binding site has been the subject of several such studies [4-91. We have shown that native puromycin can itself be used as a specific photoaffmity label [4,5]. To investigate the question of to what extent the labeling results obtained in this work were dependent on the special photochemical reaction(s) leading to incorporation, we have begun a program to repeat the photoaffinity labeling experiment with several different photolabile derivatives of puromycin.…”

Immunoelectron microscopic localization of the site of photo-induced affinity labeling of the small ribosomal subunit with puromycin.

“…Photoaffinity labeling of the ribosome has proved to be especially instructive in attempts to relate structure and function in this complex assemblage of proteins and nucleic acids (5)(6)(7)(8)(9). The photoinduced incorporation of puromycin into each of the ribosomal subunits in particular has been shown to be biochemically significant and highly specific (10)(11)(12)(13).…”

“…The procedures for photolytic incorporation of [8-3H1-puromycin (Amersham) and the analysis of photolabeled products have also been described (10)(11)(12)(13) Antibodies were induced in rabbits by toe-pad injection of a conjugate of bovine serum albumin and N6;N6-dimethyladenosine (22,25). A globulin fraction was isolated by ammonium sulfate precipitation (0-40% saturation fraction) and gel filtration on Ultrogel ACA22 (LKB).…”