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Conformational alterations in the ermC transcript in vivo during induction
1Department of Molecular Biology, University of Wisconsin, Madison 53706.
Abstract:
ermC is an inducible antibiotic resistance gene from Staphylococcus aureus, one of several whose expression is regulated at the level of mRNA secondary structure. During induction of ermC, the inhibition of a ribosome active in translation of a short leader peptide by low levels of antibiotic belonging to the macrolide-lincosamide-streptogramin b family is believed to cause a rearrangement in mRNA secondary structure. The resultant conformational isomerization unmasks the methylase ribosome binding site and initiator Met codon, causing increased translation of the ermC transcript. Expression of ermC can also be demonstrated in Bacillus subtilis carrying plasmid pE194. To probe the ermC transcript in vivo during induction, ermC was transferred to B. subtilis by transformation and the resultant transformants were treated with dimethyl sulfate which reacts with N-1 of adenine and N-3 of cytosine residues in a manner that is sensitive to secondary structure. The bases modified in vivo were detected by primer extension with reverse transcriptase using total cellular RNA as template and a complementary ermC-specific oligonucleotide as primer. Physical evidence was obtained for the secondary structural rearrangements predicted by the ermC regulatory model. Additionally, physical evidence was obtained demonstrating that during induction, the stalled ribosome protects codons 9 and 10 of the leader peptide from modification by dimethyl sulfate, in agreement with genetic data obtained previously that identified the integrity of codons 5-9 as critical for induction of ermC by erythromycin.
Insights
The ermC gene
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- The ermC gene confers inducible antibiotic resistance in Staphylococcus aureus.
- Its expression is regulated by mRNA secondary structure rearrangements.
- This mechanism involves ribosome stalling during translation of a leader peptide.
Purpose of the Study:
- To investigate the in vivo secondary structural changes of the ermC transcript during antibiotic induction.
- To provide physical evidence for the proposed regulatory model of ermC expression.
- To confirm the role of specific leader peptide codons in ermC induction.
Main Methods:
- The ermC gene was expressed in Bacillus subtilis.
- Dimethyl sulfate was used to probe mRNA structure in vivo.
- Primer extension with reverse transcriptase identified modified bases.
- Analysis focused on changes in mRNA secondary structure and ribosome protection sites.
Main Results:
- Physical evidence confirmed secondary structure rearrangements in the ermC transcript upon induction.
- Stalled ribosomes were shown to protect specific codons (9 and 10) of the leader peptide from chemical modification.
- These findings align with previous genetic data on critical codons for induction.
Conclusions:
- The study provides in vivo physical evidence supporting the mRNA secondary structure-based regulatory model for ermC.
- Ribosome stalling at specific leader peptide codons is a key event in ermC induction.
- This mechanism highlights a sophisticated control of antibiotic resistance gene expression.