Inactivation of Escherichia coli ribosomes by ultraviolet irradiation

Kagawa, Haruo; Fukutome, Hideo; Kawade, Yoshimi

doi:10.1016/0022-2836(67)90295-1

Cited by 24 publications

(11 citation statements)

References 8 publications

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“…Irradiation at 253.7 nm was found to inactivate ribosomes in the poly(U) assay (100% aqueous medium) via a one-hit process, in accord with earlier findings (29,43). At a ribosome concentration of 50 A260 units/ml, the half-life for activity loss was 5 min.…”

Section: Resultssupporting

confidence: 76%

“…Use of incorporation as a saturation function therefore requires that a correction be made for any effective light dose changes as a function of puromycin concentration. In our experiments, the effective dose is inversely proportional to the total absorbance (A2s3.7) of the solution (29,42). Accordingly, we corrected our incorporation data (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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

Cooperman¹,

Jaynes²,

Brunswick³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

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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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Section: Resultssupporting

confidence: 76%

Section: Resultsmentioning

confidence: 99%

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

Cooperman¹,

Jaynes²,

Brunswick³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

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“…To answer this question two groups of inactivation agents could be used: (a) chemical or enzymatic agents reacting specifically with some ribosomal components or their functional groups, and (b) physical agents, causing more or less unspecific damages following the absorption of a known amount of energy. In the latter group, both ionizing irradiation (Kucan, 1966) and ultraviolet light (Kagawa, Fukutome, and Kawade, 1967) have been shown to inactivate E. coli ribosomes. In both cases exponential decline of activity with dose was observed and the size of sensitive target was estimated.…”

Section: Introductionmentioning

confidence: 99%

“…In both cases exponential decline of activity with dose was observed and the size of sensitive target was estimated. Assuming that, the inactivation of ribosomes by ultraviolet light is solely due to photohydration of uracil residues, and taking into account the inactivation cross section of uracil residues determined in ordered structures, such as tRNA, Kagawa et al (1967) concluded that the majority of the pyrimidine bases in ribosome may be photolyzed without concomitant inactivation of the particle. On the other hand, from inactivation data obtained by gamma irradiation of lyophilized E. coli ribosomes it was estimated that one hit of gamma irradiation into the 70S ribosomes causes the inactivation (Kucan, 1966).…”

Section: Introductionmentioning

confidence: 99%

Functional Inactivation and Appearance of Breaks in RNA Chains Caused by Gamma Irradiation of Escherichia coli Ribosomes

Kućan¹,

Herak²,

Pečevsky-Kućan³

1971

Biophysical Journal

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70S ribosomes and 30S ribosomal subunits from Escherichia coli MRE 600 were exposed to gamma irradiation at -80szC. Exponential decline of activity with dose was observed when the ability of ribosomes to support the synthesis of polyphenylalanine was assayed. Irradiated ribosomes showed also an increased thermal lability. D(37) values of 2.2 MR and 4.8 MR, corresponding to radiation-sensitive molecular weights of 3.1 x 10(5) and 1.4 x 10(5), were determined for inactivation of 70S ribosomes and 30S subunits, respectively. Zone sedimentation analysis of RNA isolated from irradiated bacteria or 30S ribosomal subunits showed that at average, one chain scission occurs per four hits into ribosomal RNA. From these results it was concluded that the integrity of only a part of ribosomal proteins (the sum of their molecular weights not exceeding 1.4 x 10(5)) could be essential for the function of the 30S subunit in the polymerization of phenylalanine. This amount is smaller if the breaks in the RNA chain inactivate the ribosome.

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“…the functional activity of the ribosomal subunits (Kagawa et al, 1967;Tokimatsu et al, 1968;Yasuda and Fukutome, 1970).…”

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confidence: 99%

Evidence for photoinduced crosslinkage, in situ, of 30S ribosomal proteins to 16S rRNA

Gorelic¹

1975

Biochemistry

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The effects of ultraviolet radiation on the 30S ribosomal subunit of Escherichia coli were studied. Irradiation in aqueous solution under anaerobic conditions resulted in a dose-dependent decrease in the separability of the rRNA and protein components of the 30S ribosomal subunit in 4 M urea-3 M LiC1. The results of gel filtration studies of the irradiated ribosomes before and after treatment with pancreatic ribonuclease indicated that the decrease in separability of the ribosome components was a result of the photoinduced formation of covalent RNA-protein cross-links. The number of covalent cross-links was estimated to correspond to less than 3 per 10,000 daltons of ribosomal proteins. One-dimensional gel electrophoresis studies of the course of the photoinduced cross-linkage reaction indicated that cross-linkage of individual 30S ribosomal proteins to the 16S rRNA proceeds in two dose-dependent steps. The first step requires an input of 1 X 10(20) quanta of 253.7 -nm radiation and results in the cross-linkage of at least five ribosomal proteins to the 16S rRNA. The second step requires a total input of 2 X 10(20) quanta of 253.7 -nm radiation, and results in the cross-linkage of most of the remaining 30S ribosomal proteins to the 16S rRNA.

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Inactivation of Escherichia coli ribosomes by ultraviolet irradiation

Cited by 24 publications

References 8 publications

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

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

Functional Inactivation and Appearance of Breaks in RNA Chains Caused by Gamma Irradiation of Escherichia coli Ribosomes

Evidence for photoinduced crosslinkage, in situ, of 30S ribosomal proteins to 16S rRNA

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