Antibiotic effects on the photoinduced affinity labeling of Escherichia coli ribosomes by puromycin.

The effect of ribosomal antibiotics on the photoinduced affinity labeling of Escherichia coli ribosomes by puromycin [Cooperman, B.S., Jaynes, E.N., Brunswick, D.J., & Luddy, M.A. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1974; Jaynes, E.N. Jr., Grant, P.G., Giangrande, G., Wieder, R., & Cooperman, B.S. (1978) Biochemistry 17, 561] has been studied. Although blasticidin S, sparsomycin, lincomycin, and erythromycin are essentially without effect, major changes are seen on addition of either chloramphenicol or tetracycline. The products of photoincorporation have been characterized by one- and two-dimensional gel electrophoresis and by specific immunoprecipitation with antibodies to ribosomal proteins. In the presence of chloramphenicol, protein S14 becomes the major labeled protein. In the presence of tetracycline, L23 remains the major labeled protein, but the yield of labeled ribosomes is enormously increased, and the labeling is more specific for L23. These results are discussed in terms of the known modes of action of these antibiotics and the photoreactivity of tetracycline.     

Elongation factor-dependent affinity labeling of Escherichia coli ribosomes

Elongation factor-dependent affinity labeling of Escherichia coli ribosomes was obtained using a functional analogue of aminoacyl-tRNA. Since elongation factor Tu (EF-Tu) screens both the modified aminoacyl-tRNAs and the ribosomal complexes for active particles, only functional macromolecular complexes are examined. This approach also provides an unequivocal identification of the transfer RNA binding site from which affinity labeling occurs. N^ε-bromoacetyl-Lys-tRNA was prepared by covalently attaching an electrophilic group to the side-chain of the amino acid. This chemical modification did not interfere with function, since the ?BrAcLys-tRNA participated successfully in EF-Tu and poly(rA)-dependent binding to ribosomes, peptide bond formation, and elongation factor G (EF-G)-mediated translocation. Affinity labeling of ribosomal RNA was observed only in those incubations which contained both EF-Tu and EF-G. The crosslinking of ?BrAcLys-tRNA to 23 S rRNA was found even if fusidic acid was added to the incubation before EF-G. The dependence of the covalent reaction on EF-G demonstrates, unambiguously, that a reactive residue of 23 S rRNA is located adjacent to the 3′ end of the functionally defined P site. Similarly, the affinity labeling of proteins L13/14/15, L2, L32/33, and L24 required EF-G-dependent translocation of ?BrAcLys-tRNA into the P site. Protein L27 was alkylated following the EF-Tu-dependent binding of ?BrAcLys-tRNA to the ribosome, and the extent of affinity labeling was stimulated by the addition of EF-G to the incubation. Double-label dipeptide experiments confirmed that affinity labeling occurred from functional tRNA binding sites by demonstrating that the same ?BrAcLys-tRNA which reacted covalently with 23 S rRNA or a ribosomal protein could also participate in peptide bond formation. Finally, the ribosome affinity labeling obtained with ?BrAcLys-tRNA · EF-Tu · guanylylimidodiphosphate differed little from that obtained with ?BrAcLys-tRNA · EF-Tu · GTP. This work constitutes the first direct examination of the aminoacyl ends of the EF-Tu-dependent conformational states of the ribosomal complex, and demonstrates the potential value of functional Lys-tRNA analogues with different probes attached to the lysine side-chain.     

On the structural specificity of puromycin binding to Escherichia coli ribosomes

We have examined the structural specificity of the puromycin binding sites on the Escherichia coli ribosome that we have previously identified [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry 19, 3809-3817, and references cited therein] by examining the interactions of a series of adenine-containing compounds with these sites. We have used as measures of such interactions the inhibition of [3H]puromycin photoincorporation into ribosomal proteins from these sites, the site-specific photoincorporation of the 3H-labeled compounds themselves, and the inhibition of peptidyl transferase activity. For the first two of these measures we have made extensive use of a recently developed high-performance liquid chromatography (HPLC) method for ribosomal protein separation [Kerlavage, A. R., Weitzmann, C., Hasan, T., & Cooperman, B.S. (1983) J. Chromatogr. 266, 225-237]. We find that puromycin aminonucleoside (PANS) contains all of the structural elements necessary for specific binding to the three major puromycin binding sites, those of higher affinity leading to photoincorporation into L23 and S14 and that of lower affinity leading to photoincorporation into S7. Although tight binding to the L23 and S7 sites requires both the N6,N6-dimethyl and 3'-amino groups within PANS, only the N6,N6-dimethyl group and not the 3'-amino group is required for binding to the S14 site. Our current results reinforce our previous conclusion that photoincorporation into L23 takes place from the A' site within the peptidyl transferase center and lead us to speculate that the S14 site might be specific for the binding of modified nucleosides. They also force the conclusion that puromycin photoincorporation proceeds through its adenosyl moiety.     

Photoaffinity labeling of Escherichia coli ribosomes by an aryl azide analogue of puromycin. Evidence for the functional site specificity of labeling

The photoincorporation of p-azido[3H]puromycin [6-(dimethylamino)-9-[3'-deoxy-3'-[(p-azido-L-phenylalanyl)amino]-beta-D-ribofuranosyl]purine] into specific ribosomal proteins and ribosomal RNA [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry (preceding paper in this issue)] is decreased in the presence of puromycin, thus demonstrating that labeling is site specific. The magnitudes of the decreases in incorporation into the major labeled 50S proteins found on addition of different potential ribosome ligands parallel the abilities of these same ligands to inhibit peptidyltransferase. This result provides evidence that p-azidopuromycin photoincorporation into these proteins occurs at the peptidyltransferase center of the 50S subunit, a conclusion supported by other studies of ribosome structure and function. A striking new finding of this work is that puromycin aminonucleoside is a competitive inhibitor of puromycin in peptidyltransferase. The photoincorporation of p-azidopuromycin is accompanied by loss of ribosomal function, but photoincorporated p-azidopuromycin is not a competent peptidyl acceptor. The significance of these results is discussed. Photolabeling of 30S proteins by p-azidopuromycin apparently takes place from sites of lower puromycin affinity than that of the 50S site. The possible relationship of the major proteins labeled, S18, S7, and S14, to tRNA binding is considered.     

Stepwise binding of tylosin and erythromycin to Escherichia coli ribosomes, characterized by kinetic and footprinting analysis

Erythromycin and tylosin are 14- and 16-membered lactone ring macrolides, respectively. The current work shows by means of kinetic and chemical footprinting analysis that both antibiotics bind to Escherichia coli ribosomes in a two-step process. The first step established rapidly, involves a low-affinity binding site placed at the entrance of the exit tunnel in the large ribosomal subunit, where macrolides bind primarily through their hydrophobic portions. Subsequently, slow conformational changes mediated by the antibiotic hydrophilic portion push the drugs deeper into the tunnel, in a high-affinity site. Compared with erythromycin, tylosin shifts to the high-affinity site more rapidly, due to the interaction of the mycinose sugar of the drug with the loop of H35 in domain II of 23 S rRNA. Consistently, mutations of nucleosides U2609 and U754 implicated in the high-affinity site reduce the shift of tylosin to this site and destabilize, respectively, the final drug-ribosome complex. The weak interaction between tylosin and the ribosome is Mg2+ independent, unlike the tight binding. In contrast, both interactions between erythromycin and the ribosome are reduced by increasing concentrations of Mg2+ ions. Polyamines attenuate erythromycin affinity for the ribosome at both sequential steps of binding. In contrast, polyamines facilitate the initial binding of tylosin, but exert a detrimental, more pronounced, effect on the drug accommodation at its final position. Our results emphasize the role of the particular interactions that side chains of tylosin and erythromycin establish with 23 S rRNA, which govern the exact binding process of each drug and its response to the ionic environment.     

Proteins fro Escherichia coli ribosomes involved in the binding of erythromycin.

When 50 S subunits from Escherichia coli ribosomes were incubated with 1·3 m-LiC1 the resulting 1·3c core was inactive both with respect to peptidyltransferase activity and erythromycin binding (tested by equilibrium dialysis). Reconstitution experiments with purified proteins from the corresponding split fraction SP1·3 revealed that only L16 (reconstituted with the 1·3c core in a tenfold excess) could restore high activity in both systems.When 30 out of the 34 isolated ribosomal proteins were tested directly for binding or erythromycin, L15 was able to bind the drug, in contrast to all other proteins including L16. Total reconstitution experiments with the 50 S subunit demonstrated an absolute requirement for L15 and L16 with respect to both drug binding and peptidyltransferase activity.     

Properties of ribosomes from erythromycin resistant mutants of Escherichia coli.

Summary We have studied the in vitro properties of ribosomes from several mutants resistant to erythromycin. Mutations in three different genes may confer resistance to erythromycin. Two of them are structural genes for proteins L4 and L22 of the large subunit. The third mutation (in eryC gene) seems to affect mainly the small subunit. The mechanism of action of the antibiotic may involve both subunits.     

Affinity labeling at the A-site of Escherichia coli ribosomes by a non-hydrolyzable gamma-amide analog of GTP

gamma-Amides of GTP and affinity and photoaffinity derivatives of gamma-amides of GTP: gamma-anilide of GTP, gamma-(4-azido)anilide of GTP, gamma-[N-(4-azidobenzyl)-N-methyl]amide of GTP, gamma[4-N-(2-chloroethyl)-N-methylaminobenzyl]amide of GTP and gamma-[4-N-(2-oxoethyl)-N-methylaminobenzyl]amide of GTP substituted efficiently for GTP in the EF-Tu-dependent transfer of aminoacyl-tRNA to the ribosome but, in contrast to GTP, they were not hydrolyzed in this process. They represent a new class of non-hydrolyzable GTP analogs with preserved gamma-phosphodiester bond. The radioactive analog of GTP: gamma-[4-N-(2-chloroethyl)-N-methylamino[14C]benzyl]amide of GTP was used as an affinity labeling probe for the identification of components of the GTPase center formed in the EF-Tu-dependent transfer reaction of aminoacyl-tRNA to the ribosomal A-site. Within a six-component complex of poly(U)-programmed E. coli ribosomes with elongation factor Tu, Phe-tRNA(Phe) (at the A-site), tRNA(Phe) (at the P-site) and the [14C]GTP analog, mainly the ribosomal 23S RNA and to a lesser extent the ribosomal proteins L17, L21, S16, S21 and the ribosomal 16S RNA were labeled by the reagent. No significant modification of EF-Tu was detected.     

Subcloning, characterization, and affinity labeling of Escherichia coli glycinamide ribonucleotide transformylase

Glycinamide ribonucleotide transformylase (GAR TFase; EC 2.1.2.2) has been purified 70-fold to apparent homogeneity from Escherichia coli harboring an expression vector encoding the purN gene product, GAR TFase. The protein is a monomer of Mr 23,241 and catalyzes a single reaction. Steady-state kinetic parameters for the enzyme have been obtained. The structural requirements for cofactor utilization have been investigated and found to parallel those of the multifunctional avian enzyme. The enzyme was inactivated with the affinity label N10-(bromoacetyl)-5,8-dideazafolate in a stoichiometric and active-site-specific manner. The ionization state of the cofactor analogue in the enzyme-cofactor complex appears to require the dissociation of the proton at N3 of the pyrimidine within the complex.     

Photoinduced affinity labeling of the Escherichia coli ribosome puromycin site.

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.     

Photochemical affinity labeling of the macrolide binding site on the 70S E. coli ribosome

Photoactivation of the alpha, beta-unsaturated ketone-epoxide system of [3H] dihydrorosaramycin at a wavelength above 300 nm allows the covalent attachment of the antibiotic to its receptor site. The radioactivity is mainly associated to proteins L1, L5, L6, S1; as a consequence, the binding site of this type of drug could be located at the peptidyltransferase center and in between both subunits.