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 coil 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. In eukaryotes, poly(A) polymerase is essential for the maturation of mRNA, adding poly(A) tails to the 3' end of precursor RNA generated by endonucleolytic cleavage (1). Neither the specific role of poly(A) tail nor its mechanism of formation is fully understood, but recent success in the cloning of the genes for bovine poly(A) polymerase (2, 3), vaccinia virus poly(A) polymerase (4), and yeast poly(A) polymerase (5) should facilitate the elucidation of the biological role of mRNA polyadenylylation. In prokaryotes, a poly(A) polymerase activity was partially purified from Escherichia coli (6), and enzyme preparations of various purities and different properties have been described (7-9), but these enzymes were not studied further because their function was obscure. However, the recent discovery of bacterial poly(A) RNA has again focused our attention on poly(A) polymerase in E. coli as the possible source of the poly(A) moieties at the 3' ends of E. coli mRNA.Studies from this laboratory showed that substantial amounts of poly(A) sequences exist in prokaryotic mRNA from Bacillus brevis (10), Bacillus subtilis (11), and E. coli (12). The presence of large amounts of poly(A) RNA in B.subtilis was confirmed by the gligo(dT)-dependent synthesis of cDNA by using reverse transcriptase (13) and made possible the construction of the first cDNA library from bacteria (14). Finally, two specific mRNAs in E. coli, namely those for the outer membrane lipoprotein (15) and tryptophan synthetase a subunit (16), were found to be extensively (40%) polyadenylylated.To study the mechanism and physiological function of bacterial mRNA polyadenylylation, we have purified a poly(A) polymerase from E. coli, identified its gene, and achieved its overexpression by inserting the gene into an expression vector. MATERIALS AND METHODSBacterial Strains and Plasmids. Frozen cells of E. coli K-12 grown in rich medium [0.5% yeast extract/4% (wt/vol) NZ amine/0.64% K2HPO4/1% glucose] were purchased from Grain Processing (Muscatine, IA). Plasmid pJL89 carrying the pcnB locus (17) was the gift ofJ. S. Parkinson (University of Utah). The expression vector pRE1, its host E. coli C600, and E. coli MZ (A c1857) (18) were the gift of P. Reddy (National Institute of Standards and Technology).Enzymes and Chemicals. Most of the restriction endonucleases were from New England BioLabs; Taq DNA polymerase was from Perkin-Elmer/...

Targeted depletion of a mitochondrial nucleotidyltransferase suggests the presence of multiple enzymes that polymerize mRNA 3′ tails in Trypanosoma brucei mitochondria

“…Several enzymes have been described to catalyze polyadenylation reactions. The canonical PAP, represented by bacterial class II (24) or eukaryotic class I (25) PAPs, mainly synthesizes homopolymeric adenosine tails. Another enzyme, polynucleotide phosphorylase, which is primarily a degradative enzyme in vivo, has been shown to possess polyadenylation activity and create heteropolymeric tails in prokaryotes, cyanobacteria, and chloroplasts of higher plants (26)(27)(28).…”

Regulation of plasmid R1 replication: PcnB and RNase E expedite the decay of the antisense RNA, CopA

“…PcnB protein [now also called PAP I (poly(A)polymerase); Cao and Sarkar, 1992] is such a protein. Mutations in pcnB were found to decrease the copy number of ColE1-type plasmids (Lopilato et al, 1986;Liu and Parkinson, 1989;March et al, 1989).…”

RNA 3′-tail synthesis in Streptomyces: in vitro and in vivo activities of RNase PH, the SCO3896 gene product and polynucleotide phosphorylase

“…E. coli cells contain at least two enzymes capable of adding poly(A) tails to the 39-ends of RNA molecules. The major polyadenylating enzyme, designated poly(A) polymerase I (PAP I, EC 2.7.7.19), was originally identified by virtue of its role in regulating plasmid copy number, and is a product of the pcnB gene (Cao & Sarkar, 1992;He et al, 1993). Mutants of E. coli lacking PAP I still retain the ability to polyadenylate RNAs, indicating that there is at least one other polyadenylating enzyme in those cells (Kalapos et al, 1994).…”