Addition of Poly(A) and Heteropolymeric 3′ Ends in Bacillus subtilis Wild-Type and Polynucleotide Phosphorylase-Deficient Strains

Polyadenylation plays a role in decay of some bacterial mRNAs, as well as in the quality control of stable RNA. In Escherichia coli, poly(A) polymerase I (PAP I) is the main polyadenylating enzyme, but the addition of 3 tails also occurs in the absence of PAP I via the synthetic activity of polynucleotide phosphorylase (PNPase). The nature of 3-tail addition in Bacillus subtilis, which lacks an identifiable PAP I homologue, was studied. Sizing of poly(A) sequences revealed a similar pattern in wild-type and PNPase-deficient strains. Sequencing of 152 cloned cDNAs, representing 3-end sequences of nontranslated and translated RNAs, revealed modified ends mostly on incomplete transcripts, which are likely to be decay intermediates. The 3-end additions consisted of either short poly(A) sequences or longer heteropolymeric ends with a mean size of about 40 nucleotides. Interestingly, multiple independent clones exhibited complex heteropolymeric ends of very similar but not identical nucleotide sequences. Similar polyadenylated and heteropolymeric ends were observed at 3 ends of RNA isolated from wild-type and pnpA mutant strains. These data demonstrated that, unlike the case of some other bacterial species and chloroplasts, PNPase of Bacillus subtilis is not the major enzyme responsible for the addition of nucleotides to RNA 3 ends. Polyadenylation is an important posttranscriptional modification of prokaryotic, eukaryotic, and organellar RNA. In Escherichia coli, polyadenylation has been shown to play a role in the process of decay, and the enzymes responsible for both polyadenylation and degradation are known (reviewed by Grunberg-Manago [15]). Poly(A) polymerase I (PAP I), an enzyme belonging to the nucleotidyltransferase family, adds poly(A) extensions to the 3 ends of mRNAs, as well as to tRNA and rRNA (22). It has been shown that such extensions aid in the 3-to-5 exoribonucleolytic degradation of RNAs, particularly for the degradation by polynucleotide phosphorylase (PNPase) of RNAs containing structured ends, such as