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A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases
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Characterization of RNase J.

Muralidharan Vandanashree1, Ankur Kumar Singh1, Balasubramanian Gopal1

  • 1Molecular Biophysics Unit, Division of Biological Sciences, Indian Institute of Science, Bangalore, India.

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|November 4, 2023
PubMed
Summary

Ribonuclease J (RNase J) is a bacterial enzyme critical for RNA processing and degradation. This study details its structure, function, and purification, offering insights into its catalytic mechanisms and regulation.

Keywords:
Catalytic mechanismFunctional assayMetal cofactorRNase J paralogsSignature motifs

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Area of Science:

  • Molecular Biology
  • Enzymology
  • Bacterial Physiology

Background:

  • Ribonuclease J (RNase J) is essential for bacterial RNA metabolism, involved in maturation and degradation.
  • It possesses unique dual endonuclease and 5'-3' exonuclease activities.

Purpose of the Study:

  • To characterize the sequence, structural, and phylogenetic features of RNase J.
  • To elucidate the functional roles and regulatory mechanisms of RNase J in diverse organisms.
  • To provide a comprehensive guide for RNase J purification and characterization.

Main Methods:

  • Bioinformatic analysis of sequence and domain architecture.
  • Structural characterization of RNase J homologs.
  • Phylogenetic analysis across different organisms.
  • Enzyme purification strategies (with/without metal cofactor).
  • Metal cofactor identification and binding affinity assays.
  • Radioactive and fluorescence-based enzyme activity assays.

Main Results:

  • RNase J exhibits conserved structural features and a distinct active site topology coordinating a metal cofactor.
  • Enzyme activity is regulated by oligomerization, metal cofactor stoichiometry, and RNA substrate phosphorylation.
  • Purification protocols and characterization methods for RNase J and its metal cofactor were established.

Conclusions:

  • RNase J is a multifunctional enzyme crucial for bacterial RNA homeostasis.
  • Understanding its structure-activity relationship and regulation provides insights into bacterial gene expression.
  • The presented methods facilitate further research into RNase J function and its potential as a therapeutic target.