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An in vitro study of the translational attenuation model of ermC regulation
Abstract:
We have used a Bacillus subtilis in vitro translation system to test the translational attenuation model for ermC regulation. The ermC gene product is known to methylate rRNA, rendering ribosomes unable to bind this antibiotic. We have shown that the induction of ermC methylase in vitro is post-transcriptional and specific for the macrolides erythromycin and oleandomycin. Erythromycin has no significant effect on the stability of the ermC transcript in vitro, and hence the post-transcriptional induction of methylase under these conditions occurs by stimulation of translation. The induction effect requires ribosomes able to bind erythromycin. By adding small proportions of unmethylated to a methylated extract in the presence of erythromycin, methylase synthesis could be induced. Conversely, when small amounts of methylated extracts were mixed with unmethylated extracts, methylase synthesis could be maintained at elevated levels in the presence of a high concentration of erythromycin. These effects were specific for the inducible ermC, were not observed with a constitutive variant, and could be explained satisfactorily by the translational attenuation model. The roles of three segments of the ermC leader in regulation were explored by probing with appropriate complementary synthetic oligodeoxynucleotides. The induction effect of erythromycin was mimicked by using an oligonucleotide that could free the ribosome binding site for methylase.
Insights
This study demonstrates that ermC gene regulation occurs post-transcriptionally, specifically through translation stimulation by macrolide antibiotics like erythromycin, supporting the translational attenuation model.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- The ermC gene encodes a methylase that confers resistance to macrolide antibiotics by modifying ribosomal RNA.
- Ribosome binding to macrolides is inhibited by rRNA methylation, a key mechanism in antibiotic resistance.
Purpose of the Study:
- To investigate the translational attenuation model for ermC gene regulation using an in vitro translation system.
- To elucidate the post-transcriptional regulatory mechanisms of ermC gene expression in response to macrolide antibiotics.
Main Methods:
- Utilized a Bacillus subtilis in vitro translation system to study ermC gene expression.
- Assessed the effect of erythromycin and oleandomycin on ermC methylase induction.
- Employed synthetic oligodeoxynucleotides to probe regulatory segments within the ermC leader sequence.
Main Results:
- Erythromycin and oleandomycin specifically induced ermC methylase in a post-transcriptional manner.
- Erythromycin's effect on ermC induction was mediated by stimulating translation, not altering transcript stability.
- Experimental manipulations with methylated and unmethylated extracts confirmed the translational attenuation model's validity.
Conclusions:
- The translational attenuation model adequately explains the post-transcriptional regulation of ermC by macrolide antibiotics.
- Ribosome binding to erythromycin is crucial for the induction of ermC methylase synthesis.
- Specific segments of the ermC leader sequence play a role in regulating translation initiation.