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Pactamycin resistance mutations in functional sites of 16 S rRNA
1Center for Pharmaceutical Biotechnology, University of Illinois, Chicago 60607-7173, USA.
Abstract:
Mutants of an archaeon Halobacterium halobium, resistant to the universal inhibitor of translation, pactamycin, were isolated. Pactamycin resistance correlated with the presence of mutations in the 16 S rRNA gene of H. halobium single rRNA operon. Three types of mutations were found in pactamycin resistant cells, A694G, C795U and C796U (Escherichia coli 16 S rRNA numeration) located distantly in rRNA primary structure but probably neighboring each other in the three-dimensional structure. Pactamycin resistance mutations either overlapped (C795U) or were located in the immediate vicinity of nucleotides protected by the drug in E. coli and H. halobium 16 S rRNA indicating that corresponding rRNA sites might be directly involved in pactamycin binding. Ribosomal functions were not affected significantly either by mutation of C795 (one of the positions protected by the P-site-bound tRNA), or by mutations of A694 and C796 (which neighbor nucleotides protected by tRNA) suggesting that tRNA-dependent protections of C795 and G693 are explained by a conformational change in the ribosome induced by the P-site-bound tRNA. A novel mode of pactamycin action is proposed suggesting that pactamycin restricts structural transitions in 16 S rRNA preventing the ribosome from adopting a functional conformation induced by tRNA binding.
Insights
Mutations in the 16S rRNA gene of Halobacterium halobium confer resistance to pactamycin. These genetic alterations suggest pactamycin inhibits ribosomal function by restricting essential structural transitions in the 16S rRNA.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Pactamycin is a universal inhibitor of translation.
- Halobacterium halobium possesses a single rRNA operon.
- Understanding drug resistance mechanisms is crucial for developing new antibiotics.
Purpose of the Study:
- To investigate the genetic basis of pactamycin resistance in Halobacterium halobium.
- To identify specific mutations in the 16S rRNA gene associated with pactamycin resistance.
- To elucidate the mechanism of pactamycin action at the molecular level.
Main Methods:
- Isolation and characterization of pactamycin-resistant Halobacterium halobium mutants.
- Sequencing of the 16S rRNA gene in resistant mutants.
- Analysis of mutation locations relative to drug-protected sites and tRNA binding sites.
Main Results:
- Pactamycin resistance correlated with mutations (A694G, C795U, C796U) in the 16S rRNA gene.
- Mutations were located near or overlapped with nucleotides protected by pactamycin.
- Ribosomal functions were largely unaffected by mutations at specific sites, suggesting indirect effects.
Conclusions:
- Specific mutations in 16S rRNA directly confer pactamycin resistance.
- Pactamycin likely binds to specific sites on the 16S rRNA.
- A novel mechanism of action is proposed where pactamycin restricts rRNA structural transitions necessary for ribosome function.