Interaction of Bacillus subtilis Polynucleotide Phosphorylase and RNase Y STRUCTURAL MAPPING AND EFFECT ON mRNA TURNOVER

Abstract Polynucleotide phosphorylase (PNPase), a 3'-to-5' phosphorolytic exoribonuclease, is thought to be the primary enzyme responsible for turnover of Bacillus subtilis mRNA. The role of PNPase in B. subtilis mRNA decay has been analyzed previously by comparison of mRNA profiles in a wild-type strain vs. a strain that is deleted for pnpA, the gene encoding PNPase. Recent studies have provided evidence for a degradosome-like complex in B. subtilis that is built around the major decay-initiating endonuclease, RNase Y, and there is ample evidence for a strong interaction between PNPase and RNase Y. The role of the PNPase-RNase Y interaction in the exonucleolytic function of PNPase needs to be clarified. We sought to construct a B. subtilis strain containing a catalytically active PNPase that could not interact with RNase Y. Mapping studies of the PNPase-RNase Y interaction were guided by a homology model of B. subtilis PNPase based on the known structure of the Escherichia coli PNPase in complex with an RNase E peptide. Mutations in B. subtilis residues predicted to be involved in RNase Y binding showed a loss of PNPase-RNase Y interaction. Two mRNAs whose decay is dependent on RNase Y and PNPase were examined in strains containing full-length PNPase that was either catalytically active but unable to interact with RNase Y, or catalytically inactive but able to interact with RNase Y. At least for these two mRNAs, disruption of the PNPase-RNase Y interaction did not appear to affect mRNA turnover.