Modulation of RNase E activity by alternative RNA binding sites

Area of Science:

• Molecular Biology
• Biochemistry
• Microbiology

Background:

• Endoribonuclease E (RNase E) is crucial for RNA processing and homeostasis in Escherichia coli.
• RNase E regulates RNA composition and balance through targeted degradation and processing.
• Understanding RNase E's regulatory mechanisms is key to deciphering gene expression control.

Purpose of the Study:

• To investigate the regulatory role of a specific RNA binding site in RNase E (residues 25-36).
• To elucidate how mutations in this site affect RNA binding affinity and enzyme activity.
• To identify alternative RNA binding sites and their influence on RNase E function.

Main Methods:

• UV crosslinking coupled with tandem mass spectrometry to identify RNA-protein interactions.
• Site-directed mutagenesis to create Y25A and Q36R RNase E variants.
• Electrophoretic mobility shift assays (EMSAs) to assess RNA-protein complex stability.
• In vivo assays to evaluate the impact of mutations on full-length RNase E activity.

Main Results:

• Tyrosine 25 (Y25) and Glutamine 36 (Q36) were identified as key residues binding to RNA.
• The Y25A mutation resulted in hypoactivity, while the Q36R mutation led to hyperactivity.
• Mutations abolished RNA binding at the uncompetitive inhibition site but altered binding at other sites.
• RNA binding to the catalytic site correlated positively with enzyme activity.

Conclusions:

• RNase E possesses at least two distinct RNA binding sites that modulate its catalytic activity.
• The identified RNA binding sites play critical roles in regulating RNase E's function in RNA processing.
• These findings provide insights into the complex regulatory network governing RNA metabolism in E. coli.