Validation of Rnase J1 Function in Escherichia coli Cell System

Abstract

Since its discovery 35 years ago, the functional significance of catalytic RNA molecules, called ribozymes, has been better manifested through the analysis of their crystal structures. Hammerhead ribozyme of a plant satellite viroid catalyses a site-specific self-cleavage reaction, driven by an acid-base catalysis, producing RNA products with 2’3’-cyclic phosphate group and 5’-hydroxyl group. In this study, hammerhead ribozyme is utilised to validate the action of RNase J1, a ribonuclease that degrades the 5’-hydroxyl transcripts involved in glmS gene regulation during cell wall biosynthesis in Bacillus subtilis. An in vivo Hammerhead ribozyme system and an inducible-RNase J1 expression is established in BL21(DE3) cells using a two-stage Lambda Red recombineering. The first stage permitted the integration of ammerhead ribozyme fused with reporter EGFP-hDHFR gene at arsB locus of BL21(DE3) genome while the second stage integrated RNase J1 gene behind an Arabinose-inducible P ARA promoter at lacZ locus. Fluorescence assay was performed to evaluate the fluorescence (EGFP-hDHFR) production in the established strains upon graded expression of RNAse J1 under different arabinose concentrations. 49% and 60% reduction of fluorescence intensity were apparent in strain with active Hammerhead ribozyme treated with 0.0012% (w/v) and 0.0037% (w/v) arabinose, respectively, implying RNase J1 degradation of the 5’-hydroxyl tagged EGFP-hDHFR mRNA produced through the ribozyme cleavage. The implication of RNase J1 activity in E. coli in this study upholds the concept of constructing a cell based screening assay for the identification of compounds that target the glmS ribozyme of B. subtilis.