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DNA-dependent RNA polymerase from Pseudomonas aeruginosa
1Department of Microbiology and Immunology, Queen's University, Kingston, Ont., Canada.
Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
|September 1, 1987
Summary
Researchers purified Pseudomonas aeruginosa DNA-dependent RNA polymerase, revealing typical eubacterial subunits and optimal activity with Mg2+. The enzyme showed stability and sensitivity to various inhibitors, providing insights into bacterial transcription.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- DNA-dependent RNA polymerase is crucial for gene transcription in all organisms.
- Pseudomonas aeruginosa is an opportunistic pathogen, and understanding its molecular mechanisms is vital.
Purpose of the Study:
- To purify and characterize the DNA-dependent RNA polymerase from Pseudomonas aeruginosa.
- To determine the subunit composition, optimal reaction conditions, and stability of the enzyme.
Main Methods:
- Purification of RNA polymerase from Pseudomonas aeruginosa.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for subunit analysis.
- Enzyme activity assays under varying conditions (divalent cations, salt concentration, pH).
Main Results:
- The purified enzyme possessed typical eubacterial RNA polymerase subunits: beta' (157,000), beta (148,000), sigma (87,000), and alpha 2 (45,000).
- Optimal activity required Mg2+ (10 mM), with Mn2+ showing partial activity, and Ca2+/Zn2+ being ineffective.
- The enzyme exhibited stability in 50% glycerol at 4°C for over 3 months and was inhibited by heparin, streptolydigin, streptovaracin, actinomycin D, and rifampicin.
Conclusions:
- The Pseudomonas aeruginosa RNA polymerase shares structural and functional similarities with other bacterial enzymes.
- The characterization provides a basis for further studies on gene regulation in this important pathogen.
- The identified inhibitors offer potential targets for antimicrobial drug development.