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MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria
Published on: February 23, 2021
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Structural and sequence requirements for the antisense RNA regulating replication of staphylococcal multiresistance
Stephen M Kwong1, Neville Firth1
1School of Biological Sciences, University of Sydney, New South Wales 2006, Australia.
Plasmid
|January 31, 2015
Summary
The replication of Staphylococcus aureus plasmid pSK41 is controlled by antisense RNA (RNAI) that inhibits rep mRNA expression. Key RNAI structures, SLRNAI-II and SLRNAI-III, are crucial for this inhibition.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- The Staphylococcus aureus plasmid pSK41 is a conjugative multiresistance plasmid.
- Plasmid replication is often regulated by antisense RNA molecules.
- The Rep protein of pSK41 is rate-limiting for replication and its expression is negatively regulated by RNAI.
Purpose of the Study:
- To verify the secondary structures of RNAI and its target rep mRNA.
- To investigate the role of specific RNAI stem-loop structures in inhibition.
- To analyze the significance of a U-turn motif in rep mRNA for RNAI-mediated inhibition.
Main Methods:
- Enzymatic probing to determine RNA secondary structures.
- In vivo analysis of RNAI-mediated inhibition.
- Northern blotting to detect mRNA processing.
Main Results:
- Predicted secondary structures of RNAI and rep mRNA were confirmed.
- Two stem-loop structures in RNAI (SLRNAI-II and SLRNAI-III) were essential for inhibitory function.
- A U-turn motif in the rep mRNA leader region was investigated for its role in inhibition.
- Evidence suggested that rep mRNA undergoes processing.
Conclusions:
- The study supports a previously proposed model for pSK41 replication control.
- Specific structural elements in both RNAI and rep mRNA are critical for regulation.
- New questions arise regarding the detailed mechanism of plasmid replication control in Staphylococcus aureus.
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