Abstract
Members of the macrolide class of antibiotics inhibit peptide elongation on the ribosome by binding close to the peptidyltransferase center and blocking the peptide exit tunnel in the large ribosomal subunit. We have studied the modes of action of the macrolides josamycin, with a 16-membered lactone ring, and erythromycin, with a 14-membered lactone ring, in a cell-free mRNA translation system with pure components from Escherichia coli. We have found that the average lifetime on the ribosome is 3 h for josamycin and less than 2 min for erythromycin and that the dissociation constants for josamycin and erythromycin binding to the ribosome are 5.5 and 11 nM, respectively. Josamycin slows down formation of the first peptide bond of a nascent peptide in an amino acid-dependent way and completely inhibits formation of the second or third peptide bond, depending on peptide sequence. Erythromycin allows formation of longer peptide chains before the onset of inhibition. Both drugs stimulate the rate constants for drop-off of peptidyl-tRNA from the ribosome. In the josamycin case, drop-off is much faster than drug dissociation, whereas these rate constants are comparable in the erythromycin case. Therefore, at a saturating drug concentration, synthesis of full-length proteins is completely shut down by josamycin but not by erythromycin. It is likely that the bacterio-toxic effects of the drugs are caused by a combination of inhibition of protein elongation, on the one hand, and depletion of the intracellular pools of aminoacyl-tRNAs available for protein synthesis by drop-off and incomplete peptidyl-tRNA hydrolase activity, on the other hand.
Highlights
Members of the macrolide class of antibiotics inhibit peptide elongation on the ribosome by binding close to the peptidyltransferase center and blocking the peptide exit tunnel in the large ribosomal subunit
Several questions have remained unanswered. (i) Are the rate constants for peptidyl-tRNA drop-off enhanced by macrolides, or are these events an indirect consequence of the inhibition of the peptidyltransferase reaction by the drugs? (ii) At which step in the elongation cycle does peptidyl-tRNA drop-off occur? (iii) Do macrolides inhibit translocation of peptidyl-tRNA from the A to P site? (iv) Can protein synthesis be resumed by the dissociation of a macrolide that has caused ribosomal stalling? (v) Are the growth inhibitory effects of macrolides because of direct inhibition of protein elongation on the ribosome, or are they an indirect effect of depletion of tRNA pools because of frequent drop-off events and insufficient intracellular peptidyl-tRNA hydrolase activity?
Josamycin and erythromycin belong to two different subclasses of the macrolide family and have been reported to inhibit protein synthesis in principally different ways [4]
Summary
GTP, ATP, and [3H]Met were from Amersham Biosciences. Putrescine, spermidine, phosphoenolpyruvate, myokinase, and nonradioactive amino acids were from Sigma. The elongation mixture, containing EF-G (1.6 M), EF-Tu (24 M), EF-Ts (0.24 M), and tRNAbulk (ϳ0.18 mM), the relevant aminoacyl-tRNA synthetases (0.1 units/l) (defined in Ref. 21), peptidyl-tRNA hydrolase (1.12 M), and amino acids (160 M each), was preincubated for 8 min at 37 °C to allow for formation of ternary complexes. Both mixtures contained ATP (1 mM), GTP (1 mM), phosphoenolpyruvate (10 mM), myokinase (3 g/ml), and pyruvate kinase (50 g/ml)
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