The mechanism of covalent reaction of bromoacetyl-phenylalanyl-transfer RNA with the peptidyl-transfer RNA binding site of the Escherichia coli ribosome

The peptidyl-tRNA analogue N-(chloroacetyl)phenylalanyl-tRNAPhe was prepared by chemical aminoacylation and prebound to the P site of Escherichia coli ribosomes in response to poly(uridylic acid). Admixture of phenylalanyl-tRNAPhe to the A site resulted in the formation of two "dipeptides", one of which was formed by displacement of chloride ion from the peptidyl-tRNA. This constitutes the first example of ribosome-mediated formation of a peptide of altered connectivity and suggests a need for revision of the current model of peptide bond formation. Also suggested by the present finding is the feasibility of utilizing tRNAs to prepare polypeptides of altered connectivity in an in vitro protein biosynthesizing system.

supporting

confidence: 88%

Ribosome-catalyzed formation of an abnormal peptide analog

Roesser¹,

Chorghade²,

Hecht³

1986

“…The position of lysine-51 carrying the fluorescence probe was chosen according to the results of Maassen et al (1984) The catalytic center of this enzyme overlaps the binding sites of a series of ribosomal proteins including LI, L2, L14, L15, L26, and L27. In fact, affinity labeling of the P site with aminoacyl-tRNA derivatives led to recognition of L2, LI 5, and L27 (L14, L26) (Pellegrini et al, 1972(Pellegrini et al, , 1974Sapori et al, 1974;Küchler & Ofengand, 1979). Proteins localized by immune electron microscopy were LI, LI5, and L27 (Lake, 1980;Stoffler et al, 1980).…”

Section: Discussionmentioning

confidence: 99%

Localization of virginiamycin S binding site on bacterial ribosome by fluorescence energy transfer

Digiambattista¹,

Thielen²,

Maassen³

et al. 1986

Virginiamycin S, a type B synergimycin inhibiting protein synthesis in bacteria, competes with erythromycin for binding to the 50S ribosomal subunits; the mechanism of action of the two antibiotics is unclear. Energy-transfer experiments between virginiamycin S (which is endowed with inherent fluorescence due to its hydroxypicolinyl moiety) and fluorescent coumarinyl derivatives of ribosomal proteins L7 and L10 have been carried out to locate the binding site of this antibiotic on the ribosome. Previous studies have indicated that two L7/L12 dimers can attach respectively to a strong binding site located on the central protuberance and to a weak binding site located on the stalk of the 50S subunits and that protein L10 is located at the base of the stalk. The distance between ribosome-bound virginiamycin S and a fluorophore located at the strong binding site of proteins L7/L12 (Lys-51 of L7) was found to be 56 (+/- 15) A. Virginiamycin S, on the other hand, was located at a distance exceeding 67 A from the weak binding site of L7/L12 dimers. A fluorophore positioned on the unique cysteine (Cys-70) of protein L10 and ribosome-bound virginiamycin S proved to be more than 60 A apart. From data available on the location of proteins L7/L12 and L10, a model is proposed, whereby the virginiamycin S binding site is placed at the base of the central protuberance of the 50S subunits, in proximity of the presumptive peptidyl transferase center. The binding sites of macrolides and lincosamides (related antibiotics of the MLS group) are expected to be very close to that of virginiamycin S.

“…Thus, LI8 is labeled by two aryl azide derivatives of peptidyl-tRNA , and the covalently bound peptidyl-tRNAs are active as peptidyl donors in the peptidyltransferase reaction, LI5 is labeled by an electrophilic derivative of Met-tRNA™'* (Czernilofsky et al, 1974) and perhaps by an electrophilic derivative of Phe-tRNAPhe (Pellegrini et al, 1974), L13 is labeled by electrophilic derivatives of both chloramphenicol (Stóffler et al, 1980) and lysyl-tRNA (Johnson & Cantor, 1980), and LI5 and LI8 have both been shown to be proteins essential for peptidyltransferase activity in single omission-reconstitution experiments (Hampl et al, 1981). L23 is nonessential in these latter experiments but apparently lies close to LI8 within the 50S subunit on the basis of both immunoelectron microscopy experiments (Stóffler et al, 1980) and cross-linking experiments showing LI8 and L23 to be cross-linked to two 50S proteins in common, L31 and L32 (Traut et al, 1980), although no L18-L23 crosslink has been described.…”

Section: Discussionmentioning

confidence: 99%

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

Nicholson¹,

Hall²,

Strycharz³

et al. 1982

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.