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Closely Related CC- and A-adding Enzymes Collaborate to Construct and Repair the 3′-Terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans

2002; Elsevier BV; Volume: 277; Issue: 50 Linguagem: Inglês

10.1074/jbc.m207527200

ISSN

1083-351X

Autores

Kozo Tomita, Alan M. Weiner,

Tópico(s)

Genomics and Phylogenetic Studies

Resumo

The 3′-terminal CCA sequence of tRNA is faithfully constructed and repaired by the CCA-adding enzyme (ATP(CTP):tRNA nucleotidyltransferase) using CTP and ATP as substrates but no nucleic acid template. Until recently, all CCA-adding enzymes from all three kingdoms appeared to be composed of a single kind of polypeptide with dual specificity for adding both CTP and ATP; however, we recently found that in Aquifex aeolicus, which lies near the deepest root of the eubacterial 16 S rRNA-based phylogenetic tree, CCA addition represents a collaboration between closely related CC-adding and A-adding enzymes (Tomita, K. and Weiner, A. M. (2001) Science 294, 1334–1336). Here we show that inSynechocystis sp. and Deinococcus radiodurans, as in A. aeolicus, CCA is added by homologous CC- and A-adding enzymes. We also find that the eubacterial CCA-, CC-, and A-adding enzymes, as well as the related eubacterial poly(A) polymerases, each fall into phylogenetically distinct groups derived from a common ancestor. Intriguingly, the Thermatoga maritima CCA-adding enzyme groups with the A-adding enzymes, suggesting that these distinct tRNA nucleotidyltransferase activities can intraconvert over evolutionary time. The 3′-terminal CCA sequence of tRNA is faithfully constructed and repaired by the CCA-adding enzyme (ATP(CTP):tRNA nucleotidyltransferase) using CTP and ATP as substrates but no nucleic acid template. Until recently, all CCA-adding enzymes from all three kingdoms appeared to be composed of a single kind of polypeptide with dual specificity for adding both CTP and ATP; however, we recently found that in Aquifex aeolicus, which lies near the deepest root of the eubacterial 16 S rRNA-based phylogenetic tree, CCA addition represents a collaboration between closely related CC-adding and A-adding enzymes (Tomita, K. and Weiner, A. M. (2001) Science 294, 1334–1336). Here we show that inSynechocystis sp. and Deinococcus radiodurans, as in A. aeolicus, CCA is added by homologous CC- and A-adding enzymes. We also find that the eubacterial CCA-, CC-, and A-adding enzymes, as well as the related eubacterial poly(A) polymerases, each fall into phylogenetically distinct groups derived from a common ancestor. Intriguingly, the Thermatoga maritima CCA-adding enzyme groups with the A-adding enzymes, suggesting that these distinct tRNA nucleotidyltransferase activities can intraconvert over evolutionary time. The 3′-terminal CCA sequence (positions 74–76 in the standard cloverleaf representation) is universally present in the tRNAs of all organisms (1Sprinzl M. Horn C. Brown M. Ioudovitch A. Steinberg S. Nucleic Acids Res. 1998; 26: 148-153Google Scholar) and is important for many aspects of gene expression. The CCA terminus is required for the aminoacylation of tRNA (2Sprinzl M. Cramer F. Prog. Nucleic Acid Res. Mol. Biol. 1979; 22: 1-69Google Scholar, 3Tamura K. Nameki N. Hasegawa T. Shimizu M. Himeno H. J. Biol. Chem. 1994; 269: 22173-22177Google Scholar) and for translation on the ribosome where the CCA sequences of the aminoacyl- and peptidyl-tRNA pair with the large ribosomal RNA near the peptidyltransferase center (4Nissen P. Hansen J. Ban N. Moore P.B. Steitz T.A. Science. 2000; 289: 920-930Google Scholar, 5Kim D.F. Green R. Mol Cell. 1999; 4: 859-864Google Scholar, 6Green R. Noller H.F. Annu. Rev. Biochem. 1997; 66: 679-716Google Scholar). In eubacteria, the CCA sequence is required for the efficient maturation of the 5′-end of tRNA by RNase P (7Guerrier-Takada C. McClain W.H. Altman S. Cell. 1984; 38: 219-224Google Scholar, 8Oh B.K. Pace N.R. Nucleic Acids Res. 1994; 22: 4087-4094Google Scholar). In eukaryotes, the CCA sequence serves as an antideterminant to block 3′-exonuclease activity (9Mohan A. Whyte S. Wang X. Nashimoto M. Levinger L. RNA (N. Y.). 1999; 5: 245-256Google Scholar), and it is essential for the export of mature tRNA from the nucleus to the cytoplasm (10Lund E. Dahlberg J.E. Science. 1998; 282: 2082-2085Google Scholar, 11Feng W. Hopper A.K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5412-5417Google Scholar). The CCA-adding enzyme (ATP(CTP):tRNA nucleotidyltransferase) builds and repairs the 3′-terminal CCA sequence of all tRNAs (12Deutscher M.P. Jacob S.T. tRNA Nucleotidyltransferase: Enzymes of Nucleic Acid Synthesis and Modification, Part II. CRC Press, Inc., Boca Raton, FL1983: 159-183Google Scholar). CCA-adding activity has been identified in all three kingdoms, suggesting conservation of function and perhaps structure throughout evolution (13Yue D. Maizels N. Weiner A.M. RNA (N. Y.). 1996; 2: 895-908Google Scholar). CCA-adding activity is essential in some eubacteria as well as in all archaea and eukaryotes where some or all tRNA genes do not encode CCA (14Aebi M. Kirchner G. Chen J.Y. Vijayraghavan U. Jacobson A. Martin N.C. Abelson J. J. Biol. Chem. 1990; 265: 16216-16220Google Scholar). Yet, even in organisms such as Escherichia coliwhere all tRNA genes do encode CCA, CCA-adding activity confers a substantial selective advantage, probably by repairing tRNAs that have been subject to errant nucleolytic attack (15Zhu L. Deutscher M.P. EMBO J. 1987; 6: 2473-2477Google Scholar). The CCA-adding enzyme belongs to the nucleotidyltransferase (NTR) 1The abbreviations used are: NTR, nucleotidyltransferase; Aa.S, shorter Aquifex aeolicusCC-adding polypeptide; Aa.L, longer A. aeolicus A-adding polypeptide; Sy.S, shorter Synechocystis sp. CC-adding polypeptide; Sy.L, longer Synechocystis sp. A-adding polypeptide; Tm, Thermatoga maritima CCA-adding enzyme; DR.1, Deinococcus radiodurans CC-adding polypeptide; DR.2, D. radiodurans A-adding polypeptide. family, a large protein superfamily that encompasses template-dependent DNA polymerases (DNA polymerase β) and template-independent RNA and DNA polymerases (poly(A) polymerase, terminal deoxynucleotidyltransferase, and CCA-adding enzymes) as well as metabolic regulators (GlnB uridylyltransferase, glutamine synthase adenylyltransferase) and antibiotic resistance factors (kanamycin nucleotidyltransferase and streptomycin adenylyltransferase) (13Yue D. Maizels N. Weiner A.M. RNA (N. Y.). 1996; 2: 895-908Google Scholar, 16Martin G. Keller W. EMBO J. 1996; 15: 2593-2603Google Scholar, 17Holm L. Sander C. Trends Biochem. Sci. 1995; 20: 345-347Google Scholar). The CCA-adding enzyme is unique among these NTRs because, unlike other the other DNA and RNA polymerases in the superfamily, it does not use a nucleic acid template, yet it faithfully constructs a defined nucleotide sequence by the addition of mononucleotides. Several models have been proposed to explain how the CCA addition could be templated by protein alone or by a tRNA/protein complex (18Deutscher M.P. J. Biol. Chem. 1972; 247: 459-468Google Scholar, 19Shi P.Y. Maizels N. Weiner A.M. EMBO J. 1998; 17: 3197-3206Google Scholar, 20Yue D. Weiner A.M. Maizels N. J. Biol. Chem. 1998; 273: 29693-29700Google Scholar, 21Hou Y.M. RNA (N. Y.). 2000; 6: 1031-1043Google Scholar, 22Li F. Wang J. Steitz T.A. J. Mol. Biol. 2000; 304: 483-492Google Scholar, 23Tomari Y. Suzuki T. Watanabe K. Ueda T. Genes Cells. 2000; 5: 689-698Google Scholar), but the detailed mechanism of the CCA addition remains unknown. Until recently, all CCA-adding enzymes characterized from all three kingdoms were composed of a single kind of polypeptide with dual specificity for the 3′-terminal addition of CTP and ATP to tRNA primers. However, we found that CCA-adding activity in Aquifex aeolicus reflects a collaboration between two closely related nucleotidyltransferases (24Tomita K. Weiner A.M. Science. 2001; 294: 1334-1336Google Scholar). The smaller polypeptide (Aa.S) is a CC-adding enzyme that adds CTP at positions 74 and 75 of a tRNA primer. The larger polypeptide (Aa.L) is an A-adding enzyme that adds a single ATP at position 76 of a tRNA primer. Remarkably, the A. aeolicus Aa.S and Aa.L polypeptides do not appear to bind to each other or to work in concert. We also found that Thermatoga maritima encodes a polypeptide that is homologous to Aa.L over its entire length, yet it has CCA- instead of A-adding activity; moreover, the homologous N-terminal halves of the T. maritimaCCA-adding enzyme and the Aa.L A-adding enzyme are dispensible for NTR activity. Here we show that the addition of CCA to tRNA is the joint responsibility of homologous CC- and A-adding enzymes in two other eubacteria, Synechocystis sp. and Deinococcus radiodurans, just as it is in A. aeolicus. A phylogenetic analysis of eubacterial CCA-, CC-, and A-adding enzymes, as well as of poly(A) polymerases, places the CC- and A-adding enzymes in distinct groups from CCA-adding enzymes and poly(A) polymerases. We discuss several different evolutionary scenarios that could explain these data. As shown in Fig. 1A,Synechocystis sp. encodes two nucleotidyltransferase homologs, designated Sy.S and Sy.L for short and long polypeptides, with accession numbers NP_442458 and NP_441479 respectively (25Kaneko T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Hirosawa M. Sugiura M. Sasamoto S. Kimura T. Hosouchi T. Matsuno A. Muraki A. Nakazaki N. Naruo K. Okumura S. Shimpo S. Takeuchi C. Wada T. Watanabe A. Yamada M. Yasuda M. Tabata S. DNA Res. 1996; 3: 109-136Google Scholar). The coding regions were amplified from Synechocystis sp. PCC 6803 genomic DNA (generous gift from T. Kaneko of the Kazusa DNA Research Institute) using PCR primer pairs Sy.SF (5′-GGGCATATGCTTTGTCCTGTGAGCCACCTG-3′) and Sy.SR (5′-CCCCTCGAGAGATTTTCCCAGTTCTTGAGCATA-3′) for Sy.S and Sy.LCF (5′-GGGCATATGCGGACCGATGTGTTACGGCAATTATTG-3′) and Sy.LCR (5′-CCCCTCGAGAACTTTGTTCGGTAAAGGATAATG-3′) for the C-terminal half of Sy.L (residues 399–942). To allow cloning of the coding region between the NdeI andXhoI sites of the pET22b(+) expression vector (Novagen), the Sy.SF and Sy.LCF primers contained an NdeI site (underlined) overlapping the start codon, and the Sy.SR and Sy.LCR primers contained a XhoI site (underlined and italicized) overlapping the natural stop codon, which was changed to a serine TCG codon. The resulting plasmids expressing Sy.S and Sy.LC are pETSy.S and pETSy.LC, respectively. Note that Sy.LC contains about 50 additional N-terminal residues beyond the region of homology with Aa.LC and Tm.C (Fig. 1 and Ref. 24Tomita K. Weiner A.M. Science. 2001; 294: 1334-1336Google Scholar); without these N-terminal residues, Sy.LC was insoluble whether it had an N-terminal or a C-terminal hexahistidine tag. As shown in Fig. 1A, Deinococcus radioduransencodes two NTR homologs, designated DR.1 and DR.2, with accession numbers NP_294707 and NP_294915 (26White O. Eisen J.A. Heidelberg J.F. Hickey E.K. Peterson J.D. Dodson R.J. Haft D.H. Gwinn M.L. Nelson W.C. Richardson D.L. Moffat K.S. Qin H. Jiang L. Pamphile W. Crosby M. Shen M. Vamathevan J.J. Lam P. McDonald L. Utterback T. Zalewski C. Makarova K.S. Aravind L. Daly M.J. Fraser C.M. et al.Science. 1999; 286: 1571-1577Google Scholar). The coding regions were amplified from genomic DNA (generous gift from S. Wolin of Yale University) using PCR primer pairs DR.1F (5′-TTTCATATGTTTCGTCGCCGTCCGCCCCTGCCGCCGTTTCCT-3′) and DR.1R (5′-TTCGGATCCGAACTTCCCGGCGTTTCCTCCGCCCA-3′) for DR.1 and DR.2F (5′-TTTCATATGGCGACCCCAGACGGCGAGCAGGTCTGG-3′) and DR.2R (5′-TTCGGATCCGAGGTCGCCTTGGGGTTTGCGCCGAGGTA3′) for DR.2. As in the Synechocystis protocol, the DR.1F and DR.1R primers contain an NdeI site (underlined) overlapping the start codon, and the DR.1R and DR.2R primers contain aBamHI site (underlined and italicized) overlapping the natural stop codon, which was changed to a serine TCG codon. PCR failed using Taq and Pfu polymerase, presumably due to the high G+C content of D. radiodurans DNA, but was successful with Herculase Hotstart DNA polymerase (Stratagene) and Me2SO. The 50-μl PCR reaction contained 1 × Herculase DNA polymerase buffer, 200 μm dNTP, 25 pmol of each primer, 2.5 units of polymerase, and 6% Me2SO. The PCR reaction conditions were one cycle at 98 °C for 3 min followed by 35 cycles consisting of 98 °C, 65 °C, and 72 °C for 45 s, 45 s, and 1 min 45 s, respectively. The PCR fragments were purified by gel electrophoresis and cloned into pCR 2.1-TOPO vector (Invitrogen), and the sequences were verified by sequencing. TheNdeI-BamHI DNA fragment of DR.1 and a partialNdeI and BamHI digest of DR.2 (which contains anNdeI site within the open reading frame) were cloned between the NdeI and BamHI sites of the pET22b(+) expression vector. The resulting plasmids expressing DR.1 and DR.2 are pETDR.1 and pETDR.2, respectively. The expression vectors were transformed into E. coli strain BL21(DE3) carrying a plasmid encoding the minor tRNAArg(argU). Transformants were grown in LB broth containing 50 μg/ml ampicillin and 25 μg/ml kanamycin at 37 °C to anA600 of 0.8, and expression was induced by the addition of 0.1 mm isopropyl β-d-thiogalactopyranoside (IPTG) for 8 h at 25 °C. The cells were lysed by sonication in buffer A (50 mm Tris-HCl (pH 7.8), 10 mm MgCl2, 0.5 m KCl, 6 mm β-mercaptoethanol, 5% glycerol, and 0.1 mm phenylmethylsulfonyl fluoride (PMSF), and the lysates were cleared by centrifugation for 30 min at 4 °C at 10,000 × g. The cleared lysates were passed over nickel nitrilotriacetic agarose columns (Qiagen), washed extensively with buffer A plus 20 mm imidazole, and the histidine-tagged proteins were eluted with buffer A containing 150 mm imidazole. The eluted proteins were dialyzed against buffer A containing 200 mm KCl and 50% glycerol, and stored at −20 °C. Protein concentrations were assayed with the Bio-Rad protein assay kit. The purity of the histidine-tagged proteins was about 90% as judged by SDS-PAGE. A. aeolicus A-adding and CC-adding enzymes were prepared as described (24Tomita K. Weiner A.M. Science. 2001; 294: 1334-1336Google Scholar). tRNA transcripts lacking CA and A (tRNA-DC and tRNA-DCC, where D is a discriminator nucleotide at position 73) were prepared by in vitrotranscription of the pmBSCCA plasmid (a kind gift of N. R. Pace) (8Oh B.K. Pace N.R. Nucleic Acids Res. 1994; 22: 4087-4094Google Scholar) linearized with FokI and BbsI, respectively, as described (27Cazenave C. Uhlenbeck O.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6972-6976Google Scholar). For tRNA lacking CCA (tRNA-D), the plasmid was mutated using the QuikChange mutagenesis kit (Stratagene) as described (24Tomita K. Weiner A.M. Science. 2001; 294: 1334-1336Google Scholar) and digested with FokI prior to in vitrotranscription. Transcripts were purified by denaturing polyacrylamide gel electrophoresis with one nucleotide resolution, excised after UV shadowing, and then eluted, ethanol precipitated, washed with cold 70% ethanol, and dried. Uniformly labeled tRNAs were prepared identically using 800 nm [α-32P]UTP (AmershamBiosciences, 3000 Ci/mmol), 25 μm UTP, and 500 μm CTP, ATP, and GTP. The desired products were located by autoradiography and then excised, eluted, and concentrated as for unlabeled products. CCA-adding assays were carried out in 50 mm glycine-NaOH (pH 8.5), 10 mmMgCl2, 25 mm KCl, 2 mmdithiothreitol, 2 μm tRNA, 15 nm enzyme, 1 mm ATP, 1 mm CTP, and 150 nm[α-32P]ATP or [α-32P]CTP (3000 Ci/mmol). After incubation for 20 min at 37 °C, reactions were stopped by adding an equal volume of stop solution (9 murea, 0.02% xylene cyanol, and 0.02% bromphenol blue) and resolved by 12% polyacrylamide gel electrophoresis in the presence of 7 m urea. The A. aeolicus CC-adding enzyme (Aa.S) and A-adding enzyme (A.aLC) were assayed identically, but at 60 °C. The following eubacterial CCA-adding, A-adding, CC-adding enzymes and poly(A) polymerase were used for the phylogenetic analysis: the eubacteria CCA-adding enzymes of E. coli (28Blattner F.R. Plunkett III, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Google Scholar), Haemophilus influenzae (29Fleischmann R.D. Adams M.D. White O. Clayton R.A. Kirkness E.F. Kerlavage A.R. Bult C.J. Tomb J.F. Dougherty B.A. Merrick J.M. McKenney K. Sutton G. FitzHugh W. Fields C. Jocayne et al.Science. 1995; 269: 496-512Google Scholar), Bacillus subtilis (30Kunst F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. Bessieres P. Bolotin A. Borchert S. Borriss R. Boursier L. Brans A. Braun M. Brignell S.C. Bron S. Brouillet S. Bruschi C.V. Caldwell B. Capuano V. Carter N.M. Choi S.K. Codani J.J. Connerton I.F. Danchin A. et al.Nature. 1997; 390: 249-256Google Scholar), Mycobacterium leprae (31Cole S.T. Eiglmeier K. Parkhill J. James K.D. Thomson N.R. Wheeler P.R. Honore N. Garnier T. Churcher C. Harris D. Mungall K. Basham D. Brown D. Chillingworth T. Connor R. Davies R.M. Devlin K. Duthoy S. Feltwell T. Fraser A. Hamlin N. Holroyd S. Hornsby T. Jagels K. Lacroix C. Maclean J. Moule S. Murphy L. Oliver K. Quail M.A. Rajandream M.A. Rutherford K.M. Rutter S. Seeger K. Simon S. Simmonds M. Skelton J. Squares R. Squares S. Stevens K. Taylor K. Whitehead S. Woodward J.R. Barrell B.G. Nature. 2001; 409: 1007-1011Google Scholar),Mycobacterium tuberculosis (32Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Barrell B.G. et al.Nature. 1998; 393: 537-544Google Scholar), and T. maritima(33Nelson K.E. Clayton R.A. Gill S.R. Gwinn M.L. Dodson R.J. Haft D.H. Hickey E.K. Peterson J.D. Nelson W.C. Ketchum K.A. McDonald L. Utterback T.R. Malek J.A. Linher K.D. Garrett M.M. Stewart A.M. Cotton M.D. Pratt M.S. Phillips C.A. Richardson D. Heidelberg J. Sutton G.G. Fleischmann R.D. Eisen J.A. Fraser C.M. et al.Nature. 1999; 399: 323-329Google Scholar); the A-adding enzymes from A. aeolicus (34Deckert G. Warren P.V. Gaasterland T. Young W.G. Lenox A.L. Graham D.E. Overbeek R. Snead M.A. Keller M. Aujay M. Huber R. Feldman R.A. Short J.M. Olsen G.J. Swanson R.V. Nature. 1998; 392: 353-358Google Scholar),Synechocystis sp. (25Kaneko T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Hirosawa M. Sugiura M. Sasamoto S. Kimura T. Hosouchi T. Matsuno A. Muraki A. Nakazaki N. Naruo K. Okumura S. Shimpo S. Takeuchi C. Wada T. Watanabe A. Yamada M. Yasuda M. Tabata S. DNA Res. 1996; 3: 109-136Google Scholar), and D. radiodurans (26White O. Eisen J.A. Heidelberg J.F. Hickey E.K. Peterson J.D. Dodson R.J. Haft D.H. Gwinn M.L. Nelson W.C. Richardson D.L. Moffat K.S. Qin H. Jiang L. Pamphile W. Crosby M. Shen M. Vamathevan J.J. Lam P. McDonald L. Utterback T. Zalewski C. Makarova K.S. Aravind L. Daly M.J. Fraser C.M. et al.Science. 1999; 286: 1571-1577Google Scholar); the CC-adding enzymes from A. aeolicus (34Deckert G. Warren P.V. Gaasterland T. Young W.G. Lenox A.L. Graham D.E. Overbeek R. Snead M.A. Keller M. Aujay M. Huber R. Feldman R.A. Short J.M. Olsen G.J. Swanson R.V. Nature. 1998; 392: 353-358Google Scholar),Synechocystis sp. (25Kaneko T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Hirosawa M. Sugiura M. Sasamoto S. Kimura T. Hosouchi T. Matsuno A. Muraki A. Nakazaki N. Naruo K. Okumura S. Shimpo S. Takeuchi C. Wada T. Watanabe A. Yamada M. Yasuda M. Tabata S. DNA Res. 1996; 3: 109-136Google Scholar), and D. radiodurans (26White O. Eisen J.A. Heidelberg J.F. Hickey E.K. Peterson J.D. Dodson R.J. Haft D.H. Gwinn M.L. Nelson W.C. Richardson D.L. Moffat K.S. Qin H. Jiang L. Pamphile W. Crosby M. Shen M. Vamathevan J.J. Lam P. McDonald L. Utterback T. Zalewski C. Makarova K.S. Aravind L. Daly M.J. Fraser C.M. et al.Science. 1999; 286: 1571-1577Google Scholar); and the poly(A) polymerases from E. coli (28Blattner F.R. Plunkett III, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Google Scholar) andH. influenza (29Fleischmann R.D. Adams M.D. White O. Clayton R.A. Kirkness E.F. Kerlavage A.R. Bult C.J. Tomb J.F. Dougherty B.A. Merrick J.M. McKenney K. Sutton G. FitzHugh W. Fields C. Jocayne et al.Science. 1995; 269: 496-512Google Scholar). The protein sequences were aligned by ClustalW (35Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Google Scholar), the alignment of the conserved 25 kDa core of the class II nucleotidyltransferase family (13Yue D. Maizels N. Weiner A.M. RNA (N. Y.). 1996; 2: 895-908Google Scholar) was edited manually, and insertions were omitted. Phylogenetic trees were constructed by the neighbor-joining method (36Saitou N. Nei M. Mol. Biol. Evol. 1987; 4: 406-425Google Scholar) based on a distance matrix with 1000 bootstrap trials. We recently found that the addition of CCA to tRNA in A. aeolicus reflects a collaborative effort between two different but closely related polypeptides, one that adds CC at positions 74 and 75 and another that adds A at position 76 (24). A BLAST search (37Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Google Scholar) using the A. aeolicus CC-adding and A-adding enzyme sequences revealed that Synechocystis sp. (25Kaneko T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Hirosawa M. Sugiura M. Sasamoto S. Kimura T. Hosouchi T. Matsuno A. Muraki A. Nakazaki N. Naruo K. Okumura S. Shimpo S. Takeuchi C. Wada T. Watanabe A. Yamada M. Yasuda M. Tabata S. DNA Res. 1996; 3: 109-136Google Scholar) encodes two NTRs designated Sy.S and Sy.L (accession numbers NP_442458 and NP_441479 respectively). Sy.S is homologous to the A. aeolicusCC-adding enzyme (Aa.S), and Sy.L is homologous to the A. aeolicus A-adding enzyme (Aa.S). Sy.L also possesses the same N-terminal 44-kDa extension of unknown function seen in the A. aeolicus A-adding enzyme (Aa.L) and T. maritimaCCA-adding enzyme (Tm) (Fig. 1). A BLAST search using the A. aeolicus CC-adding polypeptide (Aa.S) revealed that the genome of D. radiodurans (26White O. Eisen J.A. Heidelberg J.F. Hickey E.K. Peterson J.D. Dodson R.J. Haft D.H. Gwinn M.L. Nelson W.C. Richardson D.L. Moffat K.S. Qin H. Jiang L. Pamphile W. Crosby M. Shen M. Vamathevan J.J. Lam P. McDonald L. Utterback T. Zalewski C. Makarova K.S. Aravind L. Daly M.J. Fraser C.M. et al.Science. 1999; 286: 1571-1577Google Scholar) encodes an A.aS homolog designated DR.1 (accession number NP_294915). Amino acid identity between DR.1 and Aa.S is high over the entire length of the proteins. A BLAST search for homologs of the A. aeolicus A-adding enzyme (Aa.L) did not reveal any full-length homologs in D. radiodurans, but rather a smaller homolog, designated DR.2, corresponding to the C-terminal half of Aa.L (Aa.LC) (accession number NP_294707). Thus, three eubacteria (Synechocystis sp., A. aeolicus, and T. maritima) have NTR homologs with homologous N-terminal extensions of unknown function (Sy.L, Aa.L, and Tm), but D. radiodurans does not. These data indicate that the Synechocystis Sy.S andDeinococcus DR.1 polypeptides are most closely related toA. aeolicus CC-adding enzyme, and theSynechocystis Sy.L and Deinococcus DR.2 polypeptides most closely related to the A. aeolicusA-adding enzyme. The implication is that CCA-adding activity inSynechocystis sp. and D. radiodurans, as inA. aeolicus, may be the joint responsibility of two distinct but related polypeptides, although the N-terminal extension found in the Aa.L and Tm homologs is absent from DR.2. Synechocyctis Sy.S and the C-terminal half of Sy.L (Sy.LC; amino acid residues 399–942, where Thr-399 is changed to Met) were expressed in E. coli as hexahistidine-tagged proteins (Fig. 2A). The recombinant polypeptides were assayed in the presence of both ATP and CTP using tRNA substrates lacking CCA, CA, or A (tRNA-D, tRNA-DC, and tRNA-DCC, respectively, where D is discriminator base at position 73). Sy.S adds one or more CMPs to tRNA-D and tRNA-DC, but not to tRNA-DCC, and does not add AMP to any tRNA substrate (Fig. 2B, lanes 1–3). Sy.LC adds AMP to tRNA-DCC, but not to tRNA-D or tRNA-DC, and does not add CMP to any tRNA substrate (Fig. 2B,lanes 4–6). These data suggest that Sy.S and Sy.LC are likely to be CC-adding and A-adding enzymes, respectively. Indeed, reconstitution of the CCA-adding activity was observed when Sy.S and Sy.LC were combined (Fig. 2B, lanes 7–9), as demonstrated previously for the A. aeolicus polypeptides A.aLC and Aa.S (ref. 24Tomita K. Weiner A.M. Science. 2001; 294: 1334-1336Google Scholar; see also Fig. 2C). When the assays were performed in the presence of all four ribonucleotide triphosphates, only one of which was labeled, Sy.S added only CMP to tRNA-D and tRNA-DC, Sy.LC added only AMP to tRNA-DCC, and neither Sy.S nor Sy.LC added GMP or UMP to any tRNA substrate (Fig. 2D). We conclude that CCA-adding activity in Synechocystis is divided between two closely related polypeptides with different activity and specificity, just as in A. aeolicus. TheD. radiodurans DR.1 and DR.2 polypeptides were expressed as C-terminally hexahistidine-tagged polypeptides in E. coli(Fig. 3A) and assayed using the same three tRNA substrates as for the Synechocystispolypeptides. DR.1 adds one or more CMPs to tRNA-D and tRNA-DC, but not to tRNA-DCC, and does not add AMP to any tRNA substrate (Fig. 3B, lanes 1–3). DR.2 adds AMP to tRNA-DCC, but not to tRNA-D or tRNA-DC, and does not add CMP to any tRNA substrate (Fig. 3B, lanes 4–6). Thus DR.1 and DR.2 are likely to be CC-adding and A-adding enzymes, respectively. Moreover, as expected, CCA adding activity could be reconstituted when DR.1 and DR.2 were combined (Fig. 3B, lanes 7–9). The D. radiodurans DR.2 polypeptide lacks the N-terminal extension of unknown function found in the homologous Aa.L, Sy.L, and Tm polypeptides (Fig. 1). When the assays were performed using uniformly labeled tRNA substrates in the presence of all of four nucleotides, DR. 1 added one nucleotide to tRNA-D and two nucleotides to tRNA-DCC, DR.2 added only one nucleotide to tRNA-DCC, and together DR. 1 and DR.2 added three nucleotides to tRNA-D (Fig. 3C). DR.1 incorporates only CMP into tRNA lacking CCA and CA, whereas DR.2 incorporates only AMP into tRNA lacking 3′-terminal A (Fig. 3D). These are the same results obtained for the A. aeolicus Aa.S and Aa.LC polypeptides (24Tomita K. Weiner A.M. Science. 2001; 294: 1334-1336Google Scholar) and theSynechocystis Sy.S and Sy.LC polypeptides (Fig. 2,B–D). We conclude that the D. radiodurans CCA-adding activity is also divided between two closely related polypeptides of different activity and specificity as in A. aeolicus and Synechocystis sp. Having found that CCA-adding activity inSynechocystis sp. and D. radiodurans represents a collaboration between two distinct but related polypeptides (Figs. 2and 3) just as in A. aeolicus (24Tomita K. Weiner A.M. Science. 2001; 294: 1334-1336Google Scholar), we wanted to understand the phylogenetic relationship between eubacterial CCA-, CC-, and A-adding enzymes, as well as the related eubacterial poly(A) polymerases. All known CCA-adding enzymes belong to the nucleotidyltransferase superfamily, which can be divided into Class I and Class II enzymes (13Yue D. Maizels N. Weiner A.M. RNA (N. Y.). 1996; 2: 895-908Google Scholar). The archaeal CCA-adding enzymes (Class I) share a highly homologous 45-kDa core, and the eubacterial and eukaryotic CCA-adding enzymes (Class II) share a highly homologous 25-kDa core; however, Class I and Class II enzymes exhibit little obvious homology with each other outside of the immediate vicinity of the nucleotidyltransferase active site signature (17Holm L. Sander C. Trends Biochem. Sci. 1995; 20: 345-347Google Scholar). The prokaryotic poly(A) polymerases belong to Class II and share the 25-kDa core of the CCA-adding enzymes (13Yue D. Maizels N. Weiner A.M. RNA (N. Y.). 1996; 2: 895-908Google Scholar); the eukaryotic poly(A) polymerases belong to Class I (13Yue D. Maizels N. Weiner A.M. RNA (N. Y.). 1996; 2: 895-908Google Scholar) and exhibit very modest homology with the archaeal CCA-adding enzymes over roughly 25 kDa of the 45-kDa core. 2H.-D. Cho, C. Verlinde, and A. M. Weiner, manuscript in preparation. To avoid needless complication, we compared only Class II enzymes. Because it is not yet possible to distinguish Class II CCA-adding enzymes from poly(A) polymerases based solely on sequence analysis (38Raynal L.C. Krisch H.M. Carpousis A.J. J. Bacteriol. 1998; 180: 6276-6282Google Scholar), we included only those enzymes whose enzymatic activities had been experimentally demonstrated or could be confidently assumed. These are the H. influenzae CCA-adding enzyme and poly(A) polymerase, both of which are almost identical to their experimentally characterized E. coli counterparts (21Hou Y.M. RNA (N. Y.). 2000; 6: 1031-1043Google Scholar, 23Tomari Y. Suzuki T. Watanabe K. Ueda T. Genes Cells. 2000; 5: 689-698Google Scholar,39Kalapos M.P. Cao G.J. Kushner S.R. Sarkar N. Biochem. Biophys. Res. Commun. 1994; 198: 459-465Google Scholar), the B. subtilis and M. leprae CCA-adding enzymes (38Raynal L.C. Krisch H.M. Carpousis A.J. J. Bacteriol. 1998; 180: 6276-6282Google Scholar), and the CCA-adding enzyme from M. tuberculosis, which is almost identical to M. lepraeenzyme (31Cole S.T. Eiglmeier K. Parkhill J. James K.D. Thomson N.R. Wheeler P.R. Honore N. Garnier T. Churcher C. Harris D. Mungall K. Basham D. Brown D. Chillingworth T. Connor R. Davies R.M. Devlin K. Duthoy S. Feltwell T. Fraser A. Hamlin N. Holroyd S. Hornsby T. Jagels K. Lacroix C. Maclean J. Moule S. Murphy L. Oliver K. Quail M.A. Rajandream M.A. Rutherford K.M. Rutter S. Seeger K. Simon S. Simmonds M. Skelton J. Squares R. Squares S. Stevens K. Taylor K. Whitehead S. Woodward J.R. Barrell B.G. Nature. 2001; 409: 1007-1011Google Scholar). A multiple alignment of the 25-kDa core regions of these Class II enzymes, including the active site signature, is shown in Fig. 4. As shown in Fig. 5, eubacterial nucleotidyltransferases are divided into four main groups (Groups 1–4) and two subgroups (Group 4a and 4b). The Gram-negative eubacterialE. coli and H. influenzae CCA-adding enzymes (EC-CCA and HI-CCA) form a distinct group (Group 2) from the corresponding poly(A) polymerases and Gram-positive eubacterial CCA-adding enzymes (Group 4). The Gram-positive B. subtilis,M. leprae and M. tuberculosisCCA-adding enzymes (Group 4b; BS-CCA, ML-CCA and MT-CCA) are more closely related to the Gram-negative poly(A) polymerases (Group 4a; EC-PA and HI-PA) than to the CCA-adding enzymes from Gram-negativeE. coli and H. influenzae (Group 2). Among Group 4 enzymes, the B. subtilis CCA-adding enzyme is most closely related to Gram-negative poly(A) polymerases (Group 4a), consistent with a recent biochemical study showing that the B. subtilis nucleotidyltransferase, originally predicted to be poly(A) polymerase, is in fact a CCA-adding enzyme (38Raynal L.C. Krisch H.M. Carpousis A.J. J. Bacteriol. 1998; 180: 6276-6282Google Scholar). The CC-adding enzymes (AA-CC, SY-CC, and DR-CC; Group 1) and A-adding enzymes (AA-A, SY-A, and DR-A; Group 3) group separately except for one CCA-adding enzyme (TM-CCA) that groups anomalously with the A-adding enzymes (see below for discussion). Interestingly, although the three CC-adding enzymes group with each other, as do the three A-adding enzymes, the CC- and A-adding enzymes from each organism do not group together. This suggests that CC- and A-adding enzymes do not reflect gene duplication and diversification within each lineage. Instead, the division of CCA-adding activity between CC- and A-adding enzymes may have been the primitive state, consistent with placement of A. aeolicus, D. radiodurans, and T. maritima near the deepest root of the 16 S rRNA-based phylogenetic tree (40Pace N.R. Science. 1997; 276: 734-740Google Scholar, 41Woese C.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8392-8396Google Scholar). This could also explain why the T. maritima CCA-adding enzyme groups with A-adding enzymes (Group 3) rather than with other CCA- or CC-adding enzymes; the progenitor T. maritima A-adding enzyme could have acquired CC-adding activity to become a CCA-adding enzyme, thus rendering the CC-adding enzyme redundant and subject to loss. Synechocystis does not branch as early as A. aeolicus, T. maritima, and D. radioduransin the phylogenetic tree based on rRNA (40Pace N.R. Science. 1997; 276: 734-740Google Scholar, 41Woese C.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8392-8396Google Scholar). Yet, theSynechocystis CC-adding enzyme (SY-CC in Group 1) and A-adding enzyme (SY-A in Group 3) both group with the corresponding CC- and A-adding enzymes from A. aeolicus, D. radiodurans, and T. maritima. One possible explanation is that phylogenetic analysis based on proteins including DnaJ/K, EF-Tu, and DNA polymerase I revealed a close relationship between theDeinococcus-Thermus group and cyanobacteria (42Gupta R.S. Bustard K. Falah M. Singh D. J. Bacteriol. 1997; 179: 345-357Google Scholar, 43Gupta R.S. Johari V. J. Mol. Evol. 1998; 46: 716-720Google Scholar). Thus, cyanobacterial CC- and A-adding activities may also represent the primitive state. Alternatively, A. aeolicus, D. radiodurans, and Synechocystis sp. may have independently acquired the CC- and A-adding enzymes by horizontal transfer; however, this would not explain why horizontal transfer was mainly or exclusively restricted to deeply rooted eubacteria. The CCA-adding enzymes build a defined nucleic acid sequence without using a nucleic acid template. A variety of models have been proposed to explain this remarkable behavior (18Deutscher M.P. J. Biol. Chem. 1972; 247: 459-468Google Scholar, 19Shi P.Y. Maizels N. Weiner A.M. EMBO J. 1998; 17: 3197-3206Google Scholar, 20Yue D. Weiner A.M. Maizels N. J. Biol. Chem. 1998; 273: 29693-29700Google Scholar, 21Hou Y.M. RNA (N. Y.). 2000; 6: 1031-1043Google Scholar, 22Li F. Wang J. Steitz T.A. J. Mol. Biol. 2000; 304: 483-492Google Scholar, 23Tomari Y. Suzuki T. Watanabe K. Ueda T. Genes Cells. 2000; 5: 689-698Google Scholar), but the mechanism of CCA addition remains mysterious. Moreover, although eubacterial CCA-adding enzymes and poly(A) polymerases are closely related in sequence (13Yue D. Maizels N. Weiner A.M. RNA (N. Y.). 1996; 2: 895-908Google Scholar,38Raynal L.C. Krisch H.M. Carpousis A.J. J. Bacteriol. 1998; 180: 6276-6282Google Scholar, 44Masters M. March J.B. Oliver I.R. Collins J.F. Mol. Gen. Genet. 1990; 220: 341-344Google Scholar), it is still difficult to predict the activity from sequence alone (38Raynal L.C. Krisch H.M. Carpousis A.J. J. Bacteriol. 1998; 180: 6276-6282Google Scholar, 45Reichert A.S. Thurlow D.L. Morl M. Biol. Chem. 2001; 382: 1431-1438Google Scholar). We have now found that CCA-adding activity is divided between two closely related nucleotidyltransferases inSynechocystis sp. and D. radiodurans (this paper) as well as in A. aeolicus (24Tomita K. Weiner A.M. Science. 2001; 294: 1334-1336Google Scholar). At least in A. aeolicus these two nucleotidyltransferases recognize the same face of tRNA 3K. Tomita and A. M. Weiner, unpublished observations. and work independently. Comparative biochemical and structural analysis of the related eubacterial CCA-, CC-, and A-adding enzymes, as well as eubacterial poly(A) polymerases, may help to explain the specificity of these unique enzymes and the evolutionary relationships among them. We especially thank Dr. Ikuo Ogiwara of our laboratory for valuable suggestions regarding phylogenetic analysis. We also thank Dr. T. Kaneko (Kazusa DNA Research Institute) forSynechocystis sp. DNA, Dr. S. L. Wolin (Yale University) for D. radiodurans DNA, and Dr. N. Pace for the pmBDCCA plasmid.

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