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[78] A photoactivated analog of streptomycin
660
NUCLEIC ACIDS AND RIBOSOMAL SYSTEMS
[78]
fusidic acid) are similar to those of the well known GTP analogs GMPPCP or GMPPNP. Upon irradiation of the ternary complex by visible light (incandescent lamp, 400 W) both analogs form a covalent bond only with elongation factor G, the labeling of which depends on irradiation and is inhibited by the excess of the native nueleotide. Labeling of ribosomes is insignificant, i.e., less than 15%, and nonspecific. Thus, both analogs of GTP are true photoaffinity reagents, whose site of covalent binding in the specific ternary complex is localized on elongation factor G. The preparation of these photoanalogs of GTP is simple and can be used practically without modification for the radioactive microsynthesis of other photoactivated or chemically specific affinity analogs of GTP. It cannot be concluded, however, that the several groups attached to the nucleotide may disturb its function. In our case, for example, a GTP derivative similar in structure to the ~/-(4-azidobenzyl)amide of GTP, but containing an aromatic amine, the ~/-(4-azido)anilide of GTP, is almost without inhibitory ability for the cell-free poly (U)-dependent synthesis of polyphenylalanine and does not form a ternary complex with ribosomes and elongation factor G.
[78] A P h o t o a c t i v a t e d A n a l o g o f S t r e p t o m y c i n B y ALEXANDERS. GIRSHOVICH,ELENAS. BOCHKAREVA,
and YURI A. OVCHINNIXOV Streptomycin operates at the level of translation: in the ribosomal protein-synthesizing system, it inhibits polypeptide synthesis and induces miscoding. To identify the components of the streptomycin-binding center of Escherichia coli MRE 600 ribosomes, we applied the method of affinity labeling using the photoactivated analog of streptomycin1 synthesized as described below. The method is based on the possibility of modifying the antibiotic aldehyde group without affecting its functional specificity. As a photoactivated component, we used 2-nitro-4-azidobenzoylhydrazide interacting with the aldehyde group of streptomycin to form the corresponding hydrazone. Treatment of the latter by NaB3H4 reduces the hydrazone double bond and permits introduction of a radioactive label into the analog molecule. 1A. S. Girshovich, E. S. Bochkareva, and Y. A. Ovchinnikov, Mol. Gen. Genet. 144, 205 (1976).
[78]
PHOTOACTIVATED ANALOG OF STREPTOMYCIN
661
Method of Synthesis of the Photoactivated Analog of Streptomycin Synthesis of the photoactivated component, 2-nitro-4-azidobenzoylhydrazide, is described elsewhere. ~ All the procedures are carried out in the dark or in dim daylight. NH u H~N-C-NH
0
NH II NH-C-NH~ OH
/
OH •
0
/0\
J~O
CH~,
ItC "
NO,z
NNHCO~"~_~/~-N)
~
NaB}H4
O ~ NO HCH 'j.~_~ N H N H ~ N) SCHEME 1
2-Nitro-4-azidobenzoylhydrazide, 20 t,moles, in 0.4 ml of freshly distilled dioxane is added to a solution of 10 ~moles of streptomycin in 1.3 ml of 50 mM potassium phosphate at pH 6.7, and the mixture is incubated for 3 hr at room temperature. After cooling in an ice bath, 100 t~moles of NaB3H4 (Amersham, England, specific activity about 400 Ci/ mole) were added. After 1 hr, the product is precipitated and washed with ethanol, dissolved in 10 mM potassium acetate at pH 5.0, and applied to a carboxymethyl cellulose column (CM 32, Whatman), 1 }( 5 cm. The column is washed with the same buffer to remove excess radioactivity, and the analog of streptomycin is eluted in a minimal volume with 0.5 M KC1 and stored at --20 °. The purity of the product may be determined by thin-layer chromatography on Silufol (Kavalier, Czechoslovakia) in ethanol (the RI A. S. Girshovich, V. A. Pozdnyakov, and Y. A. Ovchinnikov, this volume [77].
662
NUCLEIC ACIDS AND RIBOSOMAL SYSTEMS
[79]
of the analog was zero; of 2-nitro-4-azidobenzoylhydrazide, 0.5; of NaB~H4 and the products of its hydrolysis, 0.9-1.0). By paper electrophoresis on FN-15 (Filtrak, GDR) in 0.03 M Tris chloride at pH 7.5 (900 V, 2 hr), the analog migrated to the cathode with a relative Re value of --1 as compared to picric acid. In both systems, the radioactivity was observed as a single spot, which coincided with that stained according to Sakaguchi (the guanidine group of streptomycin) and with that visible in UV light. Upon chromatography on phosphocellulose according to Hardy et al./ the analog is eluted at 0.28 M NaC1 as a single symmetrical peak and is not precipitated by 5% TCA. The concentration of the streptomycin analog in solution was determined according to a modification of the Sakaguchi method (Tomlinson and Viswanatha 4) using native streptomycin to obtain a calibration curve. The specific activity of the analog obtained was about 50 Ci/mole. Functional Properties of the Streptomycin Photoanalog z
The photoactivated analog retains the functional activity of streptomycin by two criteria: (1) it binds only to the 30 S ribosomal subparticle, and (2) it inhibits the factor-free ("nonenzymic") p-chloromercuribenzoate-stimulated poly (U) -dependent ribosomal translation system. 5 Upon irradiation (incandescent lamp, 400 W) of the reaction mixtures of the streptomycin analog with 30 S or 50 S ribosomal subparticles taken separately, the analog covalently binds predominantly to the 30 S subparticlc. There is no covalent binding without irradiation. Therefore, we have concluded that the compound is an active photoaffinity analog of streptomycin. An analysis of components labeled with this analog permitted identification of the proteins of the 30 S ribosomal subparticle located in or near the streptomycin-binding site, i.e., proteins S 7 and S 14. 8S. J. S. Hardy, C. G. Kurland, P. Voynow, and G. Mora, Biochemistry 8, 2897 (1969). G. Tomlinson and T. Viswanatha, Anal. Biochem. 60, 15 (1974).
A. S. Spirin, 0. E. Kostiashkina, and J. Jon£k, I. Mol. Biol. 101, 553 (1976).
[79] H a l o a c y l a t e d S t r e p t o m y c i n a n d P u r o m y c i n A n a l o g s B y OLAF PONGS and ERWIN REINWALD
The binding sites of streptomycin and of puromycin on bacterial ribosomes have been probed with chemically reactive derivatives of both
NUCLEIC ACIDS AND RIBOSOMAL SYSTEMS
[78]
fusidic acid) are similar to those of the well known GTP analogs GMPPCP or GMPPNP. Upon irradiation of the ternary complex by visible light (incandescent lamp, 400 W) both analogs form a covalent bond only with elongation factor G, the labeling of which depends on irradiation and is inhibited by the excess of the native nueleotide. Labeling of ribosomes is insignificant, i.e., less than 15%, and nonspecific. Thus, both analogs of GTP are true photoaffinity reagents, whose site of covalent binding in the specific ternary complex is localized on elongation factor G. The preparation of these photoanalogs of GTP is simple and can be used practically without modification for the radioactive microsynthesis of other photoactivated or chemically specific affinity analogs of GTP. It cannot be concluded, however, that the several groups attached to the nucleotide may disturb its function. In our case, for example, a GTP derivative similar in structure to the ~/-(4-azidobenzyl)amide of GTP, but containing an aromatic amine, the ~/-(4-azido)anilide of GTP, is almost without inhibitory ability for the cell-free poly (U)-dependent synthesis of polyphenylalanine and does not form a ternary complex with ribosomes and elongation factor G.
[78] A P h o t o a c t i v a t e d A n a l o g o f S t r e p t o m y c i n B y ALEXANDERS. GIRSHOVICH,ELENAS. BOCHKAREVA,
and YURI A. OVCHINNIXOV Streptomycin operates at the level of translation: in the ribosomal protein-synthesizing system, it inhibits polypeptide synthesis and induces miscoding. To identify the components of the streptomycin-binding center of Escherichia coli MRE 600 ribosomes, we applied the method of affinity labeling using the photoactivated analog of streptomycin1 synthesized as described below. The method is based on the possibility of modifying the antibiotic aldehyde group without affecting its functional specificity. As a photoactivated component, we used 2-nitro-4-azidobenzoylhydrazide interacting with the aldehyde group of streptomycin to form the corresponding hydrazone. Treatment of the latter by NaB3H4 reduces the hydrazone double bond and permits introduction of a radioactive label into the analog molecule. 1A. S. Girshovich, E. S. Bochkareva, and Y. A. Ovchinnikov, Mol. Gen. Genet. 144, 205 (1976).
[78]
PHOTOACTIVATED ANALOG OF STREPTOMYCIN
661
Method of Synthesis of the Photoactivated Analog of Streptomycin Synthesis of the photoactivated component, 2-nitro-4-azidobenzoylhydrazide, is described elsewhere. ~ All the procedures are carried out in the dark or in dim daylight. NH u H~N-C-NH
0
NH II NH-C-NH~ OH
/
OH •
0
/0\
J~O
CH~,
ItC "
NO,z
NNHCO~"~_~/~-N)
~
NaB}H4
O ~ NO HCH 'j.~_~ N H N H ~ N) SCHEME 1
2-Nitro-4-azidobenzoylhydrazide, 20 t,moles, in 0.4 ml of freshly distilled dioxane is added to a solution of 10 ~moles of streptomycin in 1.3 ml of 50 mM potassium phosphate at pH 6.7, and the mixture is incubated for 3 hr at room temperature. After cooling in an ice bath, 100 t~moles of NaB3H4 (Amersham, England, specific activity about 400 Ci/ mole) were added. After 1 hr, the product is precipitated and washed with ethanol, dissolved in 10 mM potassium acetate at pH 5.0, and applied to a carboxymethyl cellulose column (CM 32, Whatman), 1 }( 5 cm. The column is washed with the same buffer to remove excess radioactivity, and the analog of streptomycin is eluted in a minimal volume with 0.5 M KC1 and stored at --20 °. The purity of the product may be determined by thin-layer chromatography on Silufol (Kavalier, Czechoslovakia) in ethanol (the RI A. S. Girshovich, V. A. Pozdnyakov, and Y. A. Ovchinnikov, this volume [77].
662
NUCLEIC ACIDS AND RIBOSOMAL SYSTEMS
[79]
of the analog was zero; of 2-nitro-4-azidobenzoylhydrazide, 0.5; of NaB~H4 and the products of its hydrolysis, 0.9-1.0). By paper electrophoresis on FN-15 (Filtrak, GDR) in 0.03 M Tris chloride at pH 7.5 (900 V, 2 hr), the analog migrated to the cathode with a relative Re value of --1 as compared to picric acid. In both systems, the radioactivity was observed as a single spot, which coincided with that stained according to Sakaguchi (the guanidine group of streptomycin) and with that visible in UV light. Upon chromatography on phosphocellulose according to Hardy et al./ the analog is eluted at 0.28 M NaC1 as a single symmetrical peak and is not precipitated by 5% TCA. The concentration of the streptomycin analog in solution was determined according to a modification of the Sakaguchi method (Tomlinson and Viswanatha 4) using native streptomycin to obtain a calibration curve. The specific activity of the analog obtained was about 50 Ci/mole. Functional Properties of the Streptomycin Photoanalog z
The photoactivated analog retains the functional activity of streptomycin by two criteria: (1) it binds only to the 30 S ribosomal subparticle, and (2) it inhibits the factor-free ("nonenzymic") p-chloromercuribenzoate-stimulated poly (U) -dependent ribosomal translation system. 5 Upon irradiation (incandescent lamp, 400 W) of the reaction mixtures of the streptomycin analog with 30 S or 50 S ribosomal subparticles taken separately, the analog covalently binds predominantly to the 30 S subparticlc. There is no covalent binding without irradiation. Therefore, we have concluded that the compound is an active photoaffinity analog of streptomycin. An analysis of components labeled with this analog permitted identification of the proteins of the 30 S ribosomal subparticle located in or near the streptomycin-binding site, i.e., proteins S 7 and S 14. 8S. J. S. Hardy, C. G. Kurland, P. Voynow, and G. Mora, Biochemistry 8, 2897 (1969). G. Tomlinson and T. Viswanatha, Anal. Biochem. 60, 15 (1974).
A. S. Spirin, 0. E. Kostiashkina, and J. Jon£k, I. Mol. Biol. 101, 553 (1976).
[79] H a l o a c y l a t e d S t r e p t o m y c i n a n d P u r o m y c i n A n a l o g s B y OLAF PONGS and ERWIN REINWALD
The binding sites of streptomycin and of puromycin on bacterial ribosomes have been probed with chemically reactive derivatives of both