Altering the divalent metal ion preference of RNase E.

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• RNase E is a key bacterial endoribonuclease involved in RNA processing and degradation.
• This enzyme's activity is dependent on divalent metal ions.

Purpose of the Study:

• To investigate the specific divalent metal ion requirements of RNase E in vitro and in vivo.
• To elucidate the roles of magnesium (Mg2+) and manganese (Mn2+) in RNase E enzymatic activity.

Main Methods:

• In vitro enzymatic assays using natural and oligonucleotide substrates.
• Site-directed mutagenesis of RNase E (D346C and D303C mutations).
• In vivo plasmid shuffling experiments to assess enzyme function under different ionic conditions.

Main Results:

• In vitro, RNase E shows significant activity with Mg2+ and Mn2+, preferring Mn2+ for natural RNA substrates.
• The D346C mutation abolished Mg2+-dependent activity but was restored by Mn2+.
• The D303C mutation severely impaired activity regardless of the metal ion.
• In vivo, both D303C and D346C mutations were lethal, indicating an exclusive Mg2+ requirement for RNase E function within the cell.

Conclusions:

• RNase E exhibits distinct metal ion preferences in vitro (Mn2+) versus in vivo (Mg2+).
• The D346C mutation provides a tool to study Mg2+ dependence in vivo.
• These findings highlight the complex and context-dependent ionic requirements of essential bacterial enzymes.