Interference of virginiamycin M with the initiation and the elongation of peptide chains in cell-free systems

Abstract

1. The action of virginiamycin M on the initiation and elongation steps of polypeptide synthesis in the cell-free systems of Escherichia coli has been analysed using washed ribosomal particles, phage RNA as a natural messenger and highly purified initiation and elongation factors. 2. In systems containing optimal concentrations of the initiation factor IF-1, IF-2 and IF-3 none of the reactions leading to the formation of the mRNA · 70-S · fMet-tRNA initiation complex were found to be affected by the antibiotic. Gross interference with the IF-3-dependent dissociation of 70-S ribosomes and with the recycling of IF-3 was not detected either. 3. Pronounced inhibition of fMet-tRNA binding to 70-S ribosomes by the antibiotic occurs at low concentrations of IF-2. 4. The main actions exerted by virginiamycin M at optimal concentrations of the initiation factors are the inhibitions of the following reactions: [MS2-RNA·70-S·fMet-tRNA] + [EF-Tu·alanyl-tRNA·GTP] → [MS2-RNA·70-S·fMet-tRNA·alanyl-tRNA] + EF-Tu + GDP + P i [MS2-RNA·70-S·fMet-tRNA] + puromycin→fMet-puromycin + [MS2-RNA·70-S·tRNA] 5. Virginiamycin M mediates the release of substantial amounts of fMet-alanyl-tRNA subsequent to the interaction of alanyl-tRNA with the 70-S initiation complex (without addition of EF-G). It is assumed that the detachment occurred from the A site.