The Photochemical Inactivation of Peptidyl Transferase Activity

Abstract

The photochemical oxidation of the 5 0 3 ribosomal subunit results in a rapid irreversible loss of peptidyl transferase activity. The first-order rate of inactivation occurring during the first forty minutes suggests that a single reactive group is being inactivated. The pH profile of inactivation exhibits a maximum at pH 7.5.Erythromycin at a low concentration (0.04 pmol) affords significant protection. Puromycin also exerts a protective effect but at higher concentrations. Chloramphenicol, sparsomycin and lincomycin did not exert a protective effect.The loss in catalytic activity was not accompanied by a loss in substrate binding affinity of the donor and acceptor substrates.Photo-oxidation in the presence of the anionic dye Rose Bengal effectively modifies proteins under very mild conditions and exhibits under these conditions a high degree of specificity for histidine and guanine [I]. Garvin et al. [2] have reported inactivation of Escherichiu coli 70-S ribosomes by photo-oxidation in the presence of Rose Bengal and other sensitizing dyes. Noller ef al. [3] have shown that photo-oxidation of 30-S ribosomal subunits in the presence of the anionic dye Rose Bengal leads to a rapid loss of activity in protein synthesis and that three proteins and about three histidine residues are protected from photooxidation by the presence of poly(U) and tRNA.Affinity analogues of peptidyl-tRNA [4] and chloramphenicol [5,6] have provided evidence that the proteins L16, L2, L26-27 are located at the peptidyl transferase center and are probably closely associated with each other. We report here the dye-sensitized photoinactivation of peptidyl transferase activity, some characteristics of the inactivation and the protection of peptidyl transferase activity by erythromycin and puromycin but not by sparsomycin, lincomycin or chloramphenicol. MATERIALS AND METHODS Muter ials

Photoinactivation of peptidyl transferase binding sites

“…To our knowledge, any observed inhibition of the overall peptidyl transfer reaction is accompanied by a corresponding decrease of substrate binding [ 13,20-231. Our results are in contrast to the conclusion in [6], where a loss of peptidyl transferase activity was found to be caused by photooxidation of E. coli ribosomes with Rose Bengal and no changes were found in the binding of Phe-tRNA and acPhe-tRNA to the acceptor and donor sites. They interpreted their results as a loss in the catalytic activity of peptidyl transferase without having eliminated interaction of additional ribosomal binding sites with intact molecules of aminoacyl-tRNA or peptidyl-tRNA.…”

“…Photooxidation of E. coli ribosomes was carried out as in [6]. The reaction vessels contained 9 mg ribosomes in 300 ~1 buffer (30 mM Tris-HCl (pH 7.5) 20 mM MgC12, 220 mM KCl) with eosin or Rose Bengal, at the concentration indicated, were placed in an ice bath positioned 26 cm from the condenser lens of a 500 W slide projector and irradiated for 20 min.…”

Chemical evidence for the involvement of histidyl residues in the functioning of Escherichia coli elongation factor Tu

“…'; for ribosomal peptidyl.trans. ferase center [18,19] and for aminoacyl-tRNA synthetase [20]. Histidine residue(s) might thus be generally important for the binding of any protein which in the course of protein biosynthesis forms a complex with an aminoacyl-tRNA.…”

Phenylboric acids — a new group of peptidyl transferase inhibitors

“…Photooxidation in the presence of the sensitizing dye, which modifies histidine [I 1 J, results in inactivation of peptidy1 transferase [ 12,131; treatment with ethoxyformic anhydride that acylates accessible histidine residues under specific conditions 1141, abolishes peptidyl transferase activity of ribosomes f 151. Both these treatments affect not only peptide bond formation, but also the binding of acceptor substrate without altering the properties of the donor site [ 16,171, On the basis of these observations and also in regard of the known mode of action of phenylboric acids on se&e proteases [I ] f we are inchned to believe that a histidine residue, localized in the acceptor site of the peptidyl transferase, is involved both in the binding of the acceptor substrate and in peptide bond formation.…”