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PubMed1993

Interaction between the antibiotic spiramycin and a ribosomal complex active in peptide bond formation.

The inhibition of peptide bond formation by spiramycin was studied in an in vitro system derived from Escherichia coli. Peptide bonds are formed between puromycin (S) and Ac-Phe-tRNA, which is a component of complex C, i.e., of the [Ac-Phe-tRNA-70S ribosome-poly(U)] complex, according to the puromycin reaction: C+S (Ks)<==>CS (k3)==>C'+P [Synetos, D., & Coutsogeorgopoulos, C. (1987) Biochim. Biophys. Acta 923, 275-285]. It is shown that spiramycin (A) reacts with complex C and forms the spiramycin complex C*A, which is inactive toward puromycin. C*A is the tightest complex formed between complex C and any of a number of antibiotics, such as chloramphenicol, blasticidin S, lincomycin, or sparsomycin. C*A remains stable following gel chromatography on Sephadex G-200 and sucrose gradient ultracentrifugation. Detailed kinetic study suggests that C*A is formed in a variation of a two-step mechanism in which the initial encounter complex CA is kinetically insignificant and C*A is the product of a conformational change of complex CA according to the equation, C+A (kassoc)<==>(kdissoc) C*A. The rate constants of this reaction (spiramycin reaction) are kassoc = 3.0 x 10(4) M-1 s-1 and kdissoc = 5.0 x 10(-5) s-1. Such values allow the classification of spiramycin as a slow-binding, slowly reversible inhibitor; they also lead to the calculation of an apparent overall dissociation constant equal to 1.8 nM for the C*A complex. Furthermore, they render spiramycin a useful tool in the study of antibiotic action on protein synthesis in vitro. Thus, the spiramycin reaction, in conjunction with the puromycin reaction, is applied (i) to detect a strong preincubation effect exerted by chloramphenicol and lincomycin (this effect constitutes further evidence that these two antibiotics combine with complex C as slow-binding inhibitors) and (ii) to determine the rate constant for the regeneration (k7 = 2.0 x 10(-3) s-1) of complex C from the sparsomycin complex C*I [Theocharis, D. A., & Coutsogeorgopoulos, C. (1992) Biochemistry 31, 5861-5868] according to the equation, C+I (Ki)<==>CI (k6)<==>(k7) C*I. The determination of k7 enables us to calculate the apparent association rate constant of sparsomycin, (k7/Ki') = 1.0 x 10(5) M-1 s-1, where Ki' = Ki(k7/k6 + k7). It is also shown that Ac-Phe-tRNA bound to the sparsomycin complex C*I is protected against attack by hydroxylamine.(ABSTRACT TRUNCATED AT 400 WORDS)

Anti-Bacterial Agents↗
PubMed2001

Ribosomal protein L5 helps anchor peptidyl-tRNA to the P-site in Saccharomyces cerevisiae.

Our previous demonstration that mutants of 5S rRNA called mof9 can specifically alter efficiencies of programmed ribosomal frameshifting (PRF) suggested a role for this ubiquitous molecule in the maintenance of translational reading frame, though the repetitive nature of the 5S rDNA gene (>100 copies/cell) inhibited more detailed analyses. However, given the known interactions between 5S rRNA and ribosomal protein L5 (previously called L1 or YL3) encoded by an essential, single-copy gene, we monitored the effects of a series of well-defined rpl5 mutants on PRF and virus propagation. Consistent with the mof9 results, we find that the rpl5 mutants promoted increased frameshifting efficiencies in both the -1 and +1 directions, and conferred defects in the ability of cells to propagate two endogenous viruses. Biochemical analyses demonstrated that mutant ribosomes had decreased affinities for peptidyl-tRNA. Pharmacological studies showed that sparsomycin, a peptidyltransferase inhibitor that specifically increases the binding of peptidyl-tRNA with ribosomes, was antagonistic to the frameshifting defects of the most severe mutant, and the extent of sparsomycin resistance correlated with the severity of the frameshifting defects in all of the mutants. These results provide biochemical and physiological evidence that one function of L5 is to anchor peptidyl-tRNA to the P-site. A model is presented describing how decreased affinity of ribosomes for peptidyl-tRNA can affect both -1 and +1 frameshifting, and for the effects of sparsomycin.

Alleles↗
PubMed1974

Antibiotics as probes of ribosome structure: binding of chloramphenicol and erythromycin to polyribosomes; effect of other antibiotics.

Antibiotics were used as probes of ribosome topology and function. Studies of [(14)C]chloramphenicol and [(14)C]erythromycin binding to ribosomes and polyribosomes revealed the following features. The requirement of high K(+) concentration (150 mM) for [(14)C]chloramphenicol binding to NH(4)Cl-washed ribosomes resulted from the washing procedure. Neither native 70S ribosomes nor polyribosomes require K(+) greater than 30 mM for [(14)C]chloramphenicol binding. Whereas [(14)C]chloramphenicol binds to both ribosomes and polyribosomes, [(14)C]erythromycin binds essentially only to ribosomes. After removal of peptidyl-transfer ribonucleic acid (tRNA) from polyribosomes, [(14)C]erythromycin could then be bound. The effects of a number of antibiotics on [(14)C]chloramphenicol binding to ribosomes and polyribosomes was assessed. It was found that most of the macrolides (erythromycin, carbomycin, spiramycin III, niddamycin, oleandomycin, and tylosin) and streptogramins A and B (vernamycin A, PA114A, vernamycin Balpha, and PA114B) inhibited chloramphenicol binding to NH(4)Cl-washed and native 70S ribosomes, but not to polyribosomes. After removal of peptidyl-tRNA from polyribosomes, [(14)C]chloramphenicol binding was then inhibited. In contrast, sparsomycin and althiomycin inhibited chloram-phenicol binding to polyribosomes, but not to ribosomes. After removal of peptidyl-tRNA from polyribosomes, sparsomycin and althiomycin were then ineffective. The presence of peptidyl-tRNA on polyribosomes apparently is required for binding of sparsomycin and althiomycin, but prevents binding of most macrolides and streptogramins. The lincosaminides (lincomycin and celesticetin) and methymycin (a small macrolide) inhibited [(14)C]chloramphenicol binding to NH(4)Cl-washed and native 70S ribosomes best, but also inhibited the binding to polyribosomes. The amino nucleosides and other antibiotics tested do not seem to interact strongly with the major chloramphenicol-binding site. These results provide knowledge of the interrelationships between antibiotic and substrate ribosome binding sites which should eventually contribute to a map of ribosomal topology.

Anti-Bacterial Agents↗
PubMed1976

Different susceptibility of protein synthesis to inhibitors of elongation in cell-free systems from plasma cell tumours and reticulocytes.

Plasma cells and reticulocytes are mammalian cell systems which have specialized in the synthesis of a single protein during their differentiation from one common stem cell. To study whether there is a difference in cell susceptibility at the level of elongation, dose vs. inhibition curves of sparsomycin, cycloheximide and emetine in cell-free systems with S-30 fractions from plasma cell tumours (MOPC 63, MOPC 41, RPC 20, MOPC 104 E), reticulocytes and liver were compared. The experiments revealed: (1) all the selected systems are equally sensitive to sparsomycin; (2) the susceptibility of the reticulocyte systems to cycloheximide and emetine is higher than that of the plasma cell tumours. In the dose range of 1 - 10(-7) --5 - 10(-5) M cycloheximide and 1 - 10(-6)--1 - 10(-4) M emetine the reticulocyte system is preferentially inhibited; (3) the sensitivities of all plasma cell tumours are equal; (4) the liver system is more sensitive to emetine than to cycloheximide; (5) the site of the different susceptibility to these antibiotics could be located on the ribosomes; (6) however, when the extracts of the plasma cell tumours were prepared in the presence of hemin, their susceptibility rises and is like that of reticulocytes. These results show that hemin promotes in a cell-specific manner the sensitivity to some inhibitors of protein synthesis.

Animals↗