The Escherichia coli pcnB gene promotes adenylylation of antisense RNAI of ColE1-type plasmids in vivo and degradation of RNAI decay intermediates

Abstract

Previous work has shown that RNase E-mediated cleavage of RNAI, an antisense repressor of the replication of ColE1-type plasmids, relieves repression in vivo by endonucleolytically converting RNAI to a rapidly decaying product. We report that mutations in the Escherichia coli pcnB gene result in a 10-fold prolongation of the half-life of RNAI decay intermediates and also of truncated RNAI primary transcripts lacking sites attacked by RNase E. Using Northern blotting, primer extension analysis, [32P]GTP capping of 5'-triphosphate termini, and PCR amplification methods, we show that pcnB-mediated acceleration of RNAI degradation is associated with posttranscriptional 3' addition of adenosine residues in vivo to native and processed forms of RNAI. Accumulation of antisense RNAI decay products in pcnB mutants potentially explains the reduced copy number of ColE1-type plasmids seen in the mutated bacteria.