Characterization of RNase J

Abstract

RNase J is involved in RNA maturation as well as degradation of RNA to the level of mononucleotides. This enzyme plays a vital role in maintaining intracellular RNA levels and governs different steps of the cellular metabolism in bacteria. RNase J is the first ribonuclease that was shown to have both endonuclease and 5'-3' exonuclease activity. RNase J enzymes can be identified by their characteristic sequence features and domain architecture. The quaternary structure of RNase J plays a role in regulating enzyme activity. The structure of RNase J has been characterized from several homologs. These reveal extensive overall structural similarity alongside a distinct active site topology that coordinates a metal cofactor. The metal cofactor is essential for catalytic activity. The catalytic activity of RNase J is influenced by oligomerization, the choice and stoichiometry of metal cofactors, and the 5' phosphorylation state of the RNA substrate. Here we describe the sequence and structural features of RNase J alongside phylogenetic analysis and reported functional roles in diverse organisms. We also provide a detailed purification strategy to obtain an RNase J enzyme sample with or without a metal cofactor. Different methods to identify the nature of the bound metal cofactor, the binding affinity and stoichiometry are presented. Finally, we describe enzyme assays to characterize RNase J using radioactive and fluorescence-based strategies with diverse RNA substrates.

Keywords: Catalytic mechanism; Functional assay; Metal cofactor; RNase J paralogs; Signature motifs.