Identification of the gene for an Escherichia coli poly(A) polymerase

Abstract

Many bacterial mRNAs, like those of eukaryotes, carry a polyadenylate sequence at their 3' termini, but neither the function of the bacterial poly(A) moieties nor their biosynthesis have been elucidated. To develop a genetic tool to approach the problem of bacterial poly(A) RNA, we have sought to identify the genes responsible for mRNA polyadenylylation. A poly(A) polymerase was purified to homogeneity from extracts of Escherichia coli and subjected to N-terminal sequence analysis. The 25-residue amino acid sequence obtained was used to design primers for the amplification of the corresponding coding region by the PCR from an E. coli DNA template. A 74-base-pair DNA segment was obtained that matched a region in the pcnB locus of E. coli, a gene that had originally been identified as controlling plasmid copy number [J. Lopilato, S. Bortner & J. Beckwith (1986) Mol. Gen. Genet. 205, 285-290] and was subsequently cloned and sequenced [J. Liu & J. S. Parkinson (1989) J. Bacteriol. 171, 1254-1261]. Direct evidence that the pcnB locus encodes poly(A) polymerase was provided by the observation that a bacterial strain transformed with an inducible expression vector carrying pcnB as a translational fusion produced 100-fold elevated levels of poly(A) polymerase upon induction. No increased poly(A) polymerase activity was observed in cells transformed with expression vectors carrying truncated forms of the pcnB gene. The identification of a gene encoding bacterial poly(A) polymerase opens the way for the study of the biosynthesis and function of bacterial polyadenylylated mRNA.