DEGRADATION PATHWAY OF COPA, THE ANTISENSE RNA THAT CONTROLS REPLICATION OF PLASMID R1

RNA decay in bacteria is carried out by a number of enzymes that parti cipate in the coordinated degradation of their substrates. Endo- and e xonucleolytic cleavages as well as polyadenylation are generally invol ved in determining the half-life of RNAs. Small, untranslated antisens e RNAs are suitable model systems to study decay. A study of the pathw ay of degradation of CopA, the copy number regulator RNA of plasmid R1 , is reported here. Strains carrying mutations in the genes encoding R Nase E, polynucleotide phosphorylase (PNPase), RNase II and poly(A) po lymerase I (PcnB/PAP I) - alone or in combination - were used to inves tigate degradation patterns and relative half-lives of CopA. The resul ts obtained suggest that RNase E initiates CopA decay. Both PNPase and RNase II can degrade the major 3'-cleavage product generated by RNase E. This exonucleolytic degradation is aided by PcnB, which may imply a requirement for A-tailing. RNase II can partially protect CopA's 3'- end from PNPase-dependent degradation. Other RNases are probably invol ved in decay, since in rnb/pnp double mutants, decay still occurs, alb eit at a reduced rate. Experiments using purified RNase E identified c leavage sites in CopA in the vicinity of, but not identical to, those mapped in vivo, suggesting that the cleavage site specificity of this RNase is modulated by additional proteins in the cell, A model of CopA decay is presented and discussed.