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Crystal Structures of an Archaeal Class I CCA-Adding Enzyme and Its Nucleotide Complexes

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Molecular Cell, Vol. 12, 1165–1172, November, 2003, Copyright 2003 by Cell PressCrystal Structures of an Archaeal Class ICCA-Adding Enzyme and Its Nucleotide Complexesposttranscriptional addition of the CCA sequence usingCTP and ATP as substrates (Deutscher, 1982). In thosefew eubacterial species that, like E. coli, encode thisYong Xiong,1Fang Li,1,5Jimin Wang,1Alan M. Weiner,4and Thomas A. Steitz1,2,3,*CCA triplet in their tRNA genes, the CCA-adding enzyme1Department of Molecular Biophysics and Biochemistryserves to rebuild the CCA ends of tRNAs that have been2Department of Chemistrydegraded by exonucleolytic attacks (Zhu and Deut-Yale Universityscher, 1987).3Howard Hughes Medical InstituteIn general, polynucleotide polymerases require a tem-New Haven, Connecticut 06520plate strand that specifies the sequence of nucleotides4Department of Biochemistryto be incorporated, with the notable exceptions knownSchool of Medicineto date of the poly(A) polymerase (PAP), the terminalUniversity of Washingtondeoxynucleotidyltransferase (TdT), and the CCA-addingSeattle, Washington 98195enzyme, all of which function without a nucleic acidtemplate. Among these template-independent polymer-ases, the CCA-adding enzyme is the most intriguingSummarysince its nucleotide incorporation has the highest degreeof sequence and length specificity. Also remarkable isCCA-adding enzymes catalyze the addition of CCAthe occurrence of two classes of CCA-adding enzymesonto the 3ⴕ terminus of immature tRNAs without usingthat do not show homologous regions outside of thea nucleic acid template and have been divided intocatalytic domain (Li et al., 2002), suggesting that thistwo classes based on their amino acid sequences. Weenzyme activity might have arisen twice in evolution.have determined the crystal structures of a class IThe CCA-adding enzyme belongs to the nucleotidyl-CCA-adding enzyme from Archeoglobus fulgidus (Af-transferase superfamily (NT) that is defined by an activeCCA) and its complexes with ATP, CTP, or UTP. Al-site sequence of G[SG][LIVMFY]xR[GQ]x5,6D[LIVM][DE]-though it and the class II bacterial Bacillus stearo-[CLIVMFY]3-5(Holm and Sander, 1995; Yue et al., 1996).thermophilus CCA enzyme (BstCCA) have similarThe NT family includes a strikingly diverse array of en-dimensions and domain architectures (head, neck,zymes that add nucleotides to DNA, RNA, protein, andbody, and tail), only the polymerase domain is structur-antibiotics (Holm and Sander, 1995), and has been di-ally homologous. Moreover, the relative orientation ofvided into two classes based on their sequences (Yuethe head domain with respect to the body and tailet al., 1996). The sequences of class I enzymes havedomains, which appear likely to bind tRNA, differs sig-little similarity to other enzymes within the class or tonificantly between the two enzyme classes. Unlike thethe class II enzymes outside the signature motif in theclass II BstCCA, this enzyme binds nucleotides non-catalytic domain, whereas class II enzymes share a ho-specifically in the absence of bound tRNA. The shapemologous 25 kDa N-terminal region but differ in theirand electrostatic charge distribution of the AfCCA en-C-terminal domains. Examples of Class I enzymes in-zyme suggests a model for tRNA binding that accountsclude archaeal CCA-adding enzymes, DNA polymerasefor the phosphates that are protected from chemical␤ (pol ␤), eukaryotic PAP, TdT, and kanamycin nucleoti-modification by tRNA binding to AfCCA. The structuresdyltransferases (KNT). Class II enzymes include eubac-of the AfCCA enzyme and the eukaryotic poly(A) poly-terial and eukaryotic CCA-adding enzymes and eubac-merase are very similar, implying a close evolutionaryterial PAPs (Yue et al., 1996; Martin and Keller, 1996).relationship between them.Crystal structures are known for the class I enzymes,pol ␤, KNT, TDT, and eukaryotic PAP, and for the classIntroductionII enzymes, eubacterial, and eukaryotic CCA-adding en-zymes (Pelletier et al., 1994; Sakon et al., 1993; DelarueMature tRNA molecules contain a universally conservedet al., 2002; Bard et al., 2000; Martin et al., 2000; Li et3⬘-terminal CCA sequence. This sequence plays a cru-al., 2002; Augustin et al., 2003). These structures demon-cial role in protein biosynthesis, since it is the site ofstrate that all NT family members share a homologoustRNA aminoacylation (Sprinzl and Cramer, 1979) andpolymerase domain (Li et al., 2002), and contain addi-interacts with the large subunit of the ribosome to enabletional domains that have different structures and func-peptide bond formation (Green and Noller, 1997; Nissentions.et al., 2000). Indispensable as it is, however, the CCAOver the past several decades a large body of informa-sequence is not encoded in many eubacterial and arch-tion has been obtained to illuminate the enzymatic pro-aeal tRNA genes and nearly all eukaryotic tRNA genescess of the CCA-adding enzyme. The current under-(Aebi et al., 1990). The maturation of tRNA thereforestanding of the enzymatic reaction can be summarizedrequires an essential polymerase, the CCA-adding en-as follows. (1) The nucleotide addition is catalyzed byzyme (tRNA nucleotidyltransferase) that catalyzes thea two-metal ion mechanism that is the same for all poly-merases (Steitz, 1998); (2) the enzyme recognizes pri-marily the acceptor stem and the T␺C stem-loop of the*Correspondence: [email protected] molecule (Shi et al., 1998); (3) the tRNA molecule5Present address: Harvard Medical School, Boston Children’s Hos-pital, 320 Longwood Avenue, Boston, Massachusetts 02115.does not move with respect to the enzyme during theMolecular Cell1166course of CCA addition (Shi et al., 1998); and (4) there walls and the body domain lining the bottom (Figure1B). The head domain has the five-stranded ␤ sheetis a single catalytic domain that contains sequence ho-mologous to the catalytic site of DNA pol ␤ (Holm and flanked by ␣ helices that is found in all members of theNT family. The neck domain contains mixed ␤ sheetsSander, 1995; Yue et al., 1998), and a single nucleotidebinding pocket exists for both CTP and ATP, which are and ␣ helices that are homologous to the central domainof the eukaryotic PAP (DALI Z-score of 9.8) (Holm andrecognized by a “protein template” on the enzyme (Liet al., 2002). Crystal structures have been obtained for Sander, 1998). The body domain, which is composedof a four-stranded ␤ sheet flanked by ␣ helices, is


Crystal Structures of an Archaeal Class I CCA-Adding Enzyme and Its Nucleotide Complexes

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