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Isolation and properties of Escherichia coli 23S-RNA pseudouridine 1911, 1915, 1917 synthase (RluD)
J Wrzesinski1, A Bakin, J Ofengand
1Polish Academy of Sciences, Institute of Bioorganic Chemistry, Poznan.
IUBMB Life
|November 22, 2000
Summary
The RluD enzyme modifies three pseudouridine sites in Escherichia coli 23S RNA. Natural RluD efficiently modifies all three sites, unlike expressed RluD, impacting cell growth.
Area of Science:
- Molecular Biology
- Biochemistry
- RNA Modification
Background:
- Pseudouridine (psi) residues are crucial modifications in ribosomal RNA (rRNA), particularly in the peptidyl transfer center (PTC) of the 23S rRNA in Escherichia coli.
- Five distinct psi synthases are responsible for the synthesis of nine psi residues in E. coli 23S rRNA.
- The RluD enzyme specifically synthesizes three clustered psi residues within the PTC's decoding site, and its absence significantly impairs cell growth.
Purpose of the Study:
- To isolate and characterize the native RluD enzyme responsible for pseudouridylation of E. coli 23S rRNA.
- To clone and express the rluD gene and compare the activity of the expressed enzyme with the natural RluD.
- To investigate the substrate specificity and activity of RluD, particularly its dependence on magnesium ion concentration.
Main Methods:
- Isolation of the native RluD enzyme directly from E. coli without gene amplification.
- Determination of the N-terminal sequence of RluD to facilitate gene cloning.
- Gene cloning and expression of the rluD gene in E. coli.
- In vitro characterization of the enzymatic activity of both native and expressed RluD, including site-specific modification assays and analysis of activity under varying Mg2+ concentrations.
Main Results:
- The native RluD enzyme was successfully isolated and shown to modify all three target pseudouridine sites (1911, 1915, 1917) in 23S rRNA.
- The expressed RluD enzyme, while capable of modifying some sites, did not efficiently modify the 1911 site, unlike the native enzyme.
- Natural RluD exhibited broader substrate modification activity at low magnesium concentrations compared to the RluA pseudouridine synthase.
Conclusions:
- The study highlights significant differences in the enzymatic activity and substrate specificity between the native and expressed forms of the RluD enzyme.
- The efficient modification of all three clustered pseudouridine sites by natural RluD is critical for optimal ribosome function and cell viability.
- Understanding the distinct properties of native RluD provides insights into the regulation of RNA modification and its impact on bacterial physiology.