Demonstration of erythromycin-dependent stalling of ribosomes on the ermC leader transcript.
ermC encodes a methylase that modifies 23 S rRNA, conferring resistance to macrolide-lincosamide-streptogramin B antibiotics. The expression of this gene is induced by erythromycin using a translational mechanism. We have employed the inherent RNase activity of a Bacillus subtilis S-30 extract as a probe for studying the interaction of ribosomes with ermC mRNA in the presence of antibiotics. 5' end-labeled ermC runoff transcript is a substrate for this RNase activity, while the ribosome-bound region of the RNA appears to be protected. Erythromycin- and oleandomycin-dependent protection of fragments of length 79-81 was observed during the translation of end-labeled ermC transcript. This occurs only using unmethylated (erythromycin sensitive) ribosomes. Various other antibiotics including clindomycin, tylosin, and lincomycin do not show this specific protection. These effects parallel the in vivo specificity of ermC induction. The effect of erythromycin can be abolished by using oligonucleotides complementary to regions of the ermC transcript upstream from nucleotide 71 and not by using an oligonucleotide complementary to a region of ermC downstream from that position. These results are interpretable in terms of the translational attenuation model and demonstrate that erythromycin-bound ribosomes initiate translation of the leader peptide, stall upstream from nucleotide 80 on the ermC mRNA, and thus make the ribosome-binding site for methylase message available for ribosome interaction.
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D004917:Erythromycin / Q000494:pharmacology*; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis / Q000187:drug effects; D021382:Protein Sorting Signals / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D004917:Erythromycin