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/...

We had earlier identified the pcnB locus as the gene for the major Escherichia coli poly(A) polymerase (PAP I). In this report, we describe the disruption and identification of a candidate gene for a second poly(A) polymerase (PAP II) by an experimental strategy which was based on the assumption that the viability of E. coli depends on the presence of either PAP I or PAP II. The coding region thus identified is the open reading frameJ310, located at about 87 min on the E. coli chromosome. The following lines of evidence support J310 as the gene for PAP II: (i) the deduced peptide encoded byj310 has a molecular weight of 36,300, similar to the molecular weight of 35,000 estimated by gel filtration of PAP II; (ii) the deduced J310 product is a relatively hydrophobic polypeptide with a pl of 9.4, consistent with the properties of partially purified PAP II; (iii) overexpression of J310 leads to the formation of inclusion bodies whose solubilization and renaturation yields poly(A) polymerase activity that corresponds to a 35-kDa protein as shown by enzyme blotting; and (iv) expression of a J310 fusion construct with hexahistidine at the N-terminus of the coding region allowed purification of a poly(A) polymerase fraction whose major component is a 36-kDa protein. E. coli PAP II has no significant sequence homology either to PAP I or to the viral and eukaryotic poly(A) polymerases, suggesting that the bacterial poly(A) polymerases have evolved independently. An interesting feature of the PAP II sequence is the presence of sets of two paired cysteine and histidine residues that resemble the RNA binding motifs seen in some other proteins.The recent identification of the pcnB locus (1) as the gene for the major poly(A) polymerase (ATP:polyribonucleotide adenylyltransferase, EC 2.7.7.19) of Escherichia coli (2) has opened the door to the analysis of the function and mechanism of RNA polyadenylylation in prokaryotes on the molecular level. However, although it is clear that the poly(A) polymerase (PAP I) encoded bypcnB is essential for the 3'-adenylation of the antisense RNAs that control the replication of certain plasmids (3-6), the observation that disruption of the pcnB locus by a mini-kan insertion (7) leads only to a modest reduction in the level of mRNA polyadenylylation (2) suggests that E. coli has more than one poly(A) polymerases. Indeed, we were able to identify a second poly(A) polymerase (PAP II) in extracts of E. coli with a pcnB deletion (8).To elucidate the function of polyadenylylation of mRNA in E. coli, it is essential to define the relative roles of the two poly(A) polymerases in RNA metabolism and processing. This can be accomplished only if the genes for both enzymes are identified. In this paper, we describe our successful strategy for disrupting the gene for PAP II in the absence of a functional chromosomal gene for PAP I, which has led to the identification of the previously unassigned open reading frame f310 (9) as the gene encoding PAP II. MATERIALS AND METHODSBacterial Strains and Plasm...

Although it has been known for some time that bacterial mRNA molecules carry polyadenylate moieties at their 3' ends, nothing is known about the molecular structure of bacterial poly(A) RNA. To define the polyadenylylatlon site of a specific bacterial mRNA, we took advantage of the presence of elevated levels of poly(A) RNA in cells of Escherichia coil deficient in exoribonucleases and synthesized DNA complementary to polyadenylylated lipoprotein mRNA, encoded by the lpp gene, by using avian myeloblastoslis virus reverse transcriptase and an oligo(dT)-containing primer. The 5'-terminal portion of the cDNA was amplified by the polymerase chain reaction and appropriate oligonucleotide primers, and the amplified DNA was cloned in pUC18 and subjected to nucleotide sequence analysis. Four clones were found to contain the entire 3'-terminal coding region of lpp mRNA, with poly(A) attached to either of two sites in the downstream untranslated region of the transcript. In one type of clone, the polyadenylate moiety was attached at the putative transcription termination site of lpp mRNA, whereas other clones lacked the stem-loop structure of the p-independent transcription terminator and the polyadenylate moiety was attached to the residue just preceding the terminal stem-oop of the primary transcript. A model for the polyadenylylation of bacterial mRNA is proposed in which poly(A) polymerase and exonuceases compete for the 3' ends of mRNA molecules.In recent years, much evidence has accumulated that most newly synthesized bacterial mRNA molecules resemble eukaryotic mRNA in that they contain poly(A) tracts at their 3' ends (1-9). However, it has been difficult to characterize bacterial poly(A) RNA on the molecular level, primarily due to the great instability of prokaryotic mRNA, which makes the isolation of intact mRNA much more difficult than in eukaryotes. The recent discovery that the chemical half-lives of specific mRNA species were considerably increased in Escherichia coli strains with mutations in the genes for exoribonucleases (10)(11)(12)

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The structure/function relationships of oligomycin sensitivity conferring protein (OSCP) of bovine mitochondrial ATP synthase were studied by nested deletion mutagenesis, followed by analyses of the resultant OSCPs for their ability to restore partial reactions of ATP synthesis in OSCP-depleted F1-F0 complexes. Our results indicate that, from the N-terminus of OSCP, up to 13 amino acid residues could be deleted without any effect on OSCP coupling activity. However, deletion of 16 or more residues led to a slow decline in the ability of resultant mutant forms to restore ATP synthesis. Compared to the wild-type form of OSCP, deletion mutant ND-28 (deletion of residues 1-28) is 50% as active in its ability to reconstitute ATP-Pi exchange activity. Detailed analyses of mutant ND-28 revealed that it was able to bind to the membrane segment (F0) of ATP synthase and restore oligomycin-sensitive ATPase activity in OSCP-depleted F1-F0 complexes. However, it did not bind to soluble segment F1, nor did it confer cold stability to either soluble F1 or reconstituted F1-F0 complex. On the other hand, studies on nested deletions on the C-terminal end indicate that three residues could be deleted without compromising the energy-coupling activity of OSCP. However, truncations of five or more residues caused an impairment in the ability of resultant mutant forms to restore ATP-Pi exchange activity in OSCP-depleted complexes. Mutant CD-10 (deletion of amino acids 181-190) was completely ineffective as a coupling factor. Detailed analyses of this mutant revealed that the subunit was able to bind to soluble F1 segment and confer cold stability to the enzyme but was neither able to associate with the membrane segment (F0) nor able to reconstitute high oligomycin sensitivity in depleted F1-F0 complexes. We take these data to suggest that the N-terminal end of OSCP corresponding to residues G16-N28 is essential for binding of the coupling factor to soluble F1 but not for coupling the energy of proton translocation to the synthesis of ATP; on the other hand, the carboxyl-terminal end of OSCP containing amino acids K181-M186 is important for F0-OSCP interactions as well as for the coupling of the energy of delta microH+ during the synthesis of ATP. These results suggest a model for OSCP in which the N-terminus is associated with the F1 segment and the C-terminus is associated with the F0 segment, while the central part of the polypeptide forms three or more helices constituting the stalk in the intact F1F0 enzyme.