Unpaired terminal nucleotides and 5′ monophosphorylation govern 3′ polyadenylation by Escherichia coli poly(A) polymerase I

In bacteria, most mRNAs and certain regulatory RNAs are rapidly turned over, whereas mature tRNA and ribosomal RNA are highly stable. The selective susceptibility of unstable Escherichia coli RNAs to 3' polyadenylation by the pcn8 gene product, poly(A) polymerase I (PAP I). in vivo is a key factor in their rapid degradation by 3' to 5' exonucleases. Using highly purified His-tagged recombinant PAP I, we show that differential adenylation of RNA substrates by PAP I occurs in vitro and that this capability resides in PAP I itself rather than in any ancillary protein(s). Surprisingly, the efficiency of 3' polyadenylation is affected by substrate structure at both termini; single-strand segments at either the 5' or 3' end of RNA molecules and monophosphorylation at an unpaired 5' terminus dramatically increase the rate and length of 3' poly(A) tail additions by PAP I. Our results provide a mechanistic basis for the susceptibility of certain RNAs to 3' polyadenylation. They also suggest a model of programmed RNA decay in which endonucleolytically generated RNA fragments containing single-stranded monophosphorylated 5' termini are targeted for poly(A) addition and further degradation.