In vivo oligo(A) insertions in phage MS2: role of Escherichia coli poly(A)polymerase

Previously we introduced an RNase III site into the genome of RNA phage MS2 by extending a hairpin with a perfect 18 bp long stem. One way in which th e phage escaped from being killed by RNase III cleavage was to incorporate uncoded A residues on either side of the stem. This oligo(A) stretch interr upts the perfect stem that forms the RNase III site and thus confers resist ance. In this paper we have analyzed the origin of these uncoded adenosines . The data strongly suggest that they are added by the host enzyme poly(A) polymerase. Apparently the 3'-OH created by RNase III cleavage becomes a su bstrate for poly(A) polymerase. Subsequently, MS2 replicase makes one conti guous copy from the two parts of the genome RNA. The evolutionary conversio n from RNase III sensitivity to resistance provides a large spectrum of sol utions that could be an important tool to understand what essentially const itutes an RNase III site in vivo.