Erythromycin- and Chloramphenicol-Induced Ribosomal Assembly Defects Are Secondary Effects of Protein Synthesis Inhibition (original) (raw)
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Molecular Microbiology, 2011
Inhibitors of protein synthesis cause defects in the assembly of ribosomal subunits. In response to treatment with the antibiotics erythromycin or chloramphenicol, precursors of both large and small ribosomal subunits accumulate. We have used a pulse-labelling approach to demonstrate that the accumulating subribosomal particles maturate into functional 70S ribosomes. The protein content of the precursor particles is heterogeneous and does not correspond with known assembly intermediates. Mass spectrometry indicates that production of ribosomal proteins in the presence of the antibiotics correlates with the amounts of the individual ribosomal proteins within the precursor particles. Thus, treatment of cells with chloramphenicol or erythromycin leads to an unbalanced synthesis of ribosomal proteins, providing the explanation for formation of assembly-defective particles. The operons for ribosomal proteins show a characteristic pattern of antibiotic inhibition where synthesis of the first proteins is inhibited weakly but gradually increases for the subsequent proteins in the operon. This phenomenon most likely reflects translational coupling and allows us to identify other putative coupled non-ribosomal operons in the Escherichia coli chromosome.
Erythromycin inhibition of 50S ribosomal subunit formation in Escherichia coli cells
Molecular Microbiology, 2001
The effects of erythromycin on the formation of ribosomal subunits were examined in wild-type Escherichia coli cells and in an RNase E mutant strain. Pulse±chase labelling kinetics revealed a reduced rate of 50S subunit formation in both strains compared with 30S synthesis, which was unaffected by the antibiotic. Growth of cells in the presence of [ 14 C]-erythromycin showed drug binding to 50S particles and to a 50S subunit precursor sedimenting at about 30S in sucrose gradients. Antibiotic binding to the precursor correlated with the decline in 50S formation in both strains. Erythromycin binding to the precursor showed the same 1:1 stoichiometry as binding to the 50S particle. Gel electrophoresis of rRNA from antibiotic-treated organisms revealed the presence of both 23S and 5S rRNAs in the 30S region of sucrose gradients. Hybridization with a 23S rRNAspecific probe confirmed the presence of this species of rRNA in the precursor. Eighteen 50S ribosomal proteins were associated with the precursor particle. A model is presented to account for erythromycin inhibition of 50S formation.
Properties of ribosomes from erythromycin resistant mutants of Escherichia coli
Molecular and General Genetics, 1977
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.
Molecular Pharmacology, 2003
In a cell-free system derived from Escherichia coli, it is shown that clarithromycin and roxithromycin, like their parent compound erythromycin, do not inhibit the puromycin reaction (i.e., the peptide bond formation between puromycin and AcPhe-tRNA bound at the P-site of 70S ribosomes programmed with heteropolymeric mRNA). Nevertheless, all three antibiotics compete for binding on the ribosome with tylosin, a 16-membered ring macrolide that behaves as a slow-binding, slowly reversible inhibitor of peptidyltransferase. The mutually exclusive binding of these macrolides to ribosomes is also corroborated by the fact that they protect overlapping sites in domain V of 23S rRNA from chemical modification by dimethyl sulfate. From this competition effect, detailed kinetic analysis revealed that roxithromycin or clarithromycin (A), like erythromycin, reacts rapidly with AcPhe-tRNA⅐MF-mRNA⅐70S ribosomal complex (C) to form the encounter complex CA which is then slowly isomerized to a more tight complex, termed C*A. The value of the overall dissociation constant, K A * , encompassing both steps of macrolide interaction with complex C, is 36 nM for erythromycin, 20 nM for roxithromycin, and 8 nM for clarithromycin. Because the off-rate constant of C*A complex does not significantly differ among the three macrolides, the superiority of clarithromycin as an inhibitor of translation in E. coli cells and many Gram-positive bacteria may be correlated with its greater rate of association with ribosomes.
Journal of bacteriology, 1989
The Staphylococcus aureus ermA gene, whose product confers resistance to the macrolide-lincosamide-streptogramin B family of antibiotics, is induced at the level of translation by nanomolar concentrations of erythromycin. Erythromycin also specifically stabilizes ermA transcripts, and the induced stabilization requires in-phase translation of at least one of two small leader peptides in the 5' leader region of the transcript. Erythromycin-induced mRNA stabilization was tested in three constructions in which the ermA transcript was elongated by making insertions at the ermA transcription start. Whereas mRNA downstream of the leader peptide is stabilized by erythromycin, mRNA upstream is not. In the presence of erythromycin, specific mRNA decay intermediates in both the extended ermA genes and the wild-type ermA gene were detected by both Northern blotting and S1 nuclease mapping. The 5' ends of the intermediates map to the sequences that encode each of the two ermA leader pep...
Molecular microbiology, 2006
achievements paved the way for the development of new ribosome-targeting antibiotics, some of which have already entered medical practice. The recent progress, problems and new directions of research of ribosome-targeting antibiotics are discussed in this review.
Cooperative and Antagonistic Interactions of Peptidyl-tRNA and Antibiotics with Bacterial Ribosomes
European Journal of Biochemistry, 1977
There is a single-site interaction of [mrthylene-'4C]thiamphenicol and [methylene-14C]chloramphenicol with run-off ribosomes with dissociation constants Kd = 6.8 pM and Kd = 4.6 yM respectively. Similar affinities for the antibiotics are observed in polysomes totally deprived of nascent peptides, or bearing nascent peptides on the A-site. However, two types of interaction are observed in endogenous polysomes with some ribosomes bearing nascent peptides on the P-site and others in the A-site. The lower-affinity bindings (dissociation constants Kd = 6.4 pM and Kd = 5.9 pM for thiamphenicol and chloramphenicol respectively) are due to the ribosomes bearing nascent peptides on the A-site. The higher-affinity bindings (dissociation constants Kd = 2.3 yM and Kd = 1.5 pM for thiamphenicol and chloramphenicol, respectively) are due to the ribosomes bearing nascent peptides on the P-site. Therefore binding of nascent peptides to the A-site does not affect the affinities of thiamphenicol and chloramphenicol for the ribosome. On the other hand interaction of the nascent peptides with the P-site of the ribosomes increases the affinities of both antibiotics for the ribosome. Thiamphenicol and chloramphenicol are thus good inhibitors of peptide bond formation in ribosomes and polysomes. Their affinities are increased precisely when the peptidyl-tRNA is placed in the P-site preceeding the peptide bond formation step, which is specifically blocked by the antibiotics.