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[Identification and characterization of the two promoters of plasmid pKYM]
T Hase1, T Kimura, Y Nakanishi
1Department of Microbiology, Faculty of Pharmaceutical Sciences, Kanazawa University, Japan.
Abstract:
Plasmid pKYM is a multicopy plasmid isolated from Shigella sonnei and multiples stably in Escherichia coli. The plasmid encodes Rep protein which is essential for its multiplication and synthesizes cop ribonucleic acid (RNA) which is a short RNA complementary to the 5' region of rep m-RNA. This RNA controls the copy number and the incompatibility of the plasmid. The previous analysis located the promoters of rep m-RNA RNA (PR) and cop RNA (PL) in the inc region. This report confirmed the presence of these promoters by analyzing the RNAs isolated from the cells carrying pKYM and those synthesized in vitro. The initiation sites of these transcriptions were also determined. Analysis of in vivo RNA suggested that the quantity of cop RNA whose size was about 90 nucleotides was larger than that of rep m-RNA and these RNAs easily formed RNA-RNA hybrid. The analysis also suggested that the synthesis of rep m-RNA was repressed by cop RNA and Rep protein itself.
Insights
This study identifies key RNA molecules controlling plasmid replication in Shigella sonnei. Cop RNA and Rep protein regulate plasmid copy number and stability in Escherichia coli.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Plasmid pKYM from Shigella sonnei is a multicopy plasmid that replicates stably in Escherichia coli.
- It encodes a Rep protein crucial for replication and synthesizes cop ribonucleic acid (RNA), which regulates plasmid copy number and incompatibility.
- Previous studies localized the promoters for rep messenger RNA (mRNA) and cop RNA within the inc region.
Purpose of the Study:
- To confirm the presence and determine the initiation sites of the rep mRNA and cop RNA promoters.
- To analyze the in vivo transcription and interaction of these RNAs.
- To elucidate the regulatory mechanisms of plasmid pKYM replication.
Main Methods:
- Analysis of RNAs isolated from E. coli cells harboring pKYM.
- In vitro synthesis of RNAs.
- RNA-RNA hybridization analysis.
- Determination of transcription initiation sites.
Main Results:
- Confirmed the presence of promoters for rep mRNA and cop RNA in the inc region.
- Determined the initiation sites for both transcriptions.
- Observed higher quantities of cop RNA (approx. 90 nucleotides) compared to rep mRNA in vivo.
- Demonstrated that cop RNA and Rep protein repress rep mRNA synthesis.
Conclusions:
- Cop RNA plays a significant role in regulating plasmid pKYM copy number and incompatibility.
- The interaction between cop RNA, rep mRNA, and Rep protein forms a negative feedback loop controlling plasmid replication.
- This study provides a detailed understanding of the molecular mechanisms governing pKYM plasmid stability in E. coli.