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Expression of plasmid encoded Escherichia coli 5S ribosomal ribonucleic acid in Pseudomonas putida
Abstract:
The recombinant plasmid pNRK 36, which represents the plasmids RSF 1010, a small multicopy plasmid of the incompatibility group IncQ that confers resistance to streptomycin and sulfoamide to its host cells, and pKK 223-3, which contains the try-lac (tac) promoter followed by a polylinker and a DNA segment containing the 5S rRNA (rrn B) with the ribosomal RNA transcription terminators, was employed to transform Pseudomonas putida 2440 cells. The plasmid encoded 5 S rRNA from Escherichia coli was transcribed and processed properly in P. putida cells thus demonstrating for the first time the expression of a plasmid encoded ribosomal RNA in a heterologous system. The fact that the E. coli 5 S rRNA was not incorporated into assembled ribosomes suggests that the in vivo incorporation of 5 S rRNA into the 50 S ribosomal subunit is closely linked to 23 S rRNA and/or ribosomal protein synthesis.
Insights
This study shows Escherichia coli 5S ribosomal RNA can be expressed in Pseudomonas putida. However, it was not incorporated into ribosomes, suggesting a link between 5S rRNA, 23S rRNA, and protein synthesis.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Ribosomal RNA (rRNA) is essential for protein synthesis.
- Understanding rRNA expression in heterologous systems is crucial for synthetic biology.
Purpose of the Study:
- To investigate the expression of plasmid-encoded Escherichia coli 5S rRNA in Pseudomonas putida.
- To determine if heterologous 5S rRNA can be incorporated into P. putida ribosomes.
Main Methods:
- Recombinant plasmid pNRK 36, containing E. coli 5S rRNA genes, was used to transform P. putida 2440.
- Analysis of rRNA transcription, processing, and ribosome incorporation.
Main Results:
- Plasmid-encoded E. coli 5S rRNA was successfully transcribed and processed in P. putida.
- This marks the first demonstration of plasmid-encoded rRNA expression in a heterologous bacterial system.
- The expressed E. coli 5S rRNA was not incorporated into P. putida ribosomes.
Conclusions:
- Heterologous expression of functional 5S rRNA is achievable in P. putida.
- 5S rRNA incorporation into the 50S ribosomal subunit is tightly regulated and linked to 23S rRNA and/or ribosomal protein synthesis.