PDBsum entry 1vfg

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Transferase/RNA PDB id
Protein chains
342 a.a. *
APC ×2
Waters ×79
* Residue conservation analysis
PDB id:
Name: Transferase/RNA
Title: Crystal structure of tRNA nucleotidyltransferase complexed with a primer tRNA and an incoming atp analog
Structure: RNA (75-mer). Chain: c, d. Engineered: yes. Poly a polymerase. Chain: a, b. Fragment: residues 1-390. Synonym: a-adding enzyme. Engineered: yes
Source: Synthetic: yes. Other_details: RNA was prepared by in vitro transcription with t7 RNA polymerase in thermotoga maritima. Aquifex aeolicus. Organism_taxid: 63363. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.
Biol. unit: Dimer (from PQS)
2.80Å     R-factor:   0.230     R-free:   0.286
Authors: K.Tomita,S.Fukai,R.Ishitani,T.Ueda,N.Takeuchi,D.G.Vassylyev, O.Nureki,Riken Structural Genomics/proteomics Initiative (Rsgi)
Key ref:
K.Tomita et al. (2004). Structural basis for template-independent RNA polymerization. Nature, 430, 700-704. PubMed id: 15295603 DOI: 10.1038/nature02712
13-Apr-04     Release date:   10-Aug-04    
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Protein chains
Pfam   ArchSchema ?
O66728  (O66728_AQUAE) -  Poly A polymerase
824 a.a.
342 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     RNA processing   1 term 
  Biochemical function     RNA binding     2 terms  


DOI no: 10.1038/nature02712 Nature 430:700-704 (2004)
PubMed id: 15295603  
Structural basis for template-independent RNA polymerization.
K.Tomita, S.Fukai, R.Ishitani, T.Ueda, N.Takeuchi, D.G.Vassylyev, O.Nureki.
The 3'-terminal CCA nucleotide sequence (positions 74-76) of transfer RNA is essential for amino acid attachment and interaction with the ribosome during protein synthesis. The CCA sequence is synthesized de novo and/or repaired by a template-independent RNA polymerase, 'CCA-adding enzyme', using CTP and ATP as substrates. Despite structural and biochemical studies, the mechanism by which the CCA-adding enzyme synthesizes the defined sequence without a nucleic acid template remains elusive. Here we present the crystal structure of Aquifex aeolicus CCA-adding enzyme, bound to a primer tRNA lacking the terminal adenosine and an incoming ATP analogue, at 2.8 A resolution. The enzyme enfolds the acceptor T helix of the tRNA molecule. In the catalytic pocket, C75 is adjacent to ATP, and their base moieties are stacked. The complementary pocket for recognizing C74-C75 of tRNA forms a 'protein template' for the penultimate two nucleotides, mimicking the nucleotide template used by template-dependent polymerases. These results are supported by systematic analyses of mutants. Our structure represents the 'pre-insertion' stage of selecting the incoming nucleotide and provides the structural basis for the mechanism underlying template-independent RNA polymerization.
  Selected figure(s)  
Figure 2.
Figure 2: Stereoview of the primer C74-C75 and the incoming ATP. a, [A]-Weighted simulated-annealing F[o] - F[c] omit maps contoured at 3.5 around C74-C75 and AMPcPP. The carbon atoms of Aa.LC, tRNA and AMPcPP are coloured white, pink and blue, respectively. b, Recognition of the incoming ATP. Ball-and-stick representations of tRNA C75, AMPcPP and the ATP-interacting residues are shown on the Aa.LC head and neck domains. The colouring scheme is the same as in Fig. 1. c, Recognition of the C74-C75 terminus. Ball-and-stick representations of tRNA A73-C74-C75, AMPcPP and the tRNA-interacting residues are shown. Phe 106 and the Asp105 -Arg155 pair, which are part of the 'stacking arc', are also shown in ball-and-stick representation. In b and c, hydrogen bonds are shown as dotted lines.
Figure 4.
Figure 4: Comparison of template-independent and template-dependent RNA polymerases. a, Ball-and-stick representations of tRNA, AMPcPP, the catalytic carboxylates and the ATP-interacting residues are shown on the Aa.LC head and neck domains. The colouring scheme is the same as in Fig. 2, except that the carbon atoms of Aa.LC are coloured orange. b, Ball-and-stick representations of the primer RNA, the template DNA, AMPcPP, the catalytic carboxylates and the ATP-interacting residues are shown on the O helix in the T7 RNA polymerase structure. In a and b, hydrogen bonds are shown as dotted lines.
  The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2004, 430, 700-704) copyright 2004.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20348137 A.Hoffmeier, H.Betat, A.Bluschke, R.Günther, S.Junghanns, H.J.Hofmann, and M.Mörl (2010).
Unusual evolution of a catalytic core element in CCA-adding enzymes.
  Nucleic Acids Res, 38, 4436-4447.  
21071662 B.Pan, Y.Xiong, and T.A.Steitz (2010).
How the CCA-adding enzyme selects adenine over cytosine at position 76 of tRNA.
  Science, 330, 937-940.
PDB codes: 3ouy 3ov7 3ova 3ovb 3ovs
20155482 H.Betat, C.Rammelt, and M.Mörl (2010).
tRNA nucleotidyltransferases: ancient catalysts with an unusual mechanism of polymerization.
  Cell Mol Life Sci, 67, 1447-1463.  
  20101632 Y.M.Hou (2010).
CCA addition to tRNA: implications for tRNA quality control.
  IUBMB Life, 62, 251-260.  
19270066 H.Nakayama, M.Akiyama, M.Taoka, Y.Yamauchi, Y.Nobe, H.Ishikawa, N.Takahashi, and T.Isobe (2009).
Ariadne: a database search engine for identification and chemical analysis of RNA using tandem mass spectrometry data.
  Nucleic Acids Res, 37, e47.  
19833706 K.Kuchta, L.Knizewski, L.S.Wyrwicz, L.Rychlewski, and K.Ginalski (2009).
Comprehensive classification of nucleotidyltransferase fold proteins: identification of novel families and their representatives in human.
  Nucleic Acids Res, 37, 7701-7714.  
19696158 S.Kim, C.Liu, K.Halkidis, H.B.Gamper, and Y.M.Hou (2009).
Distinct kinetic determinants for the stepwise CCA addition to tRNA.
  RNA, 15, 1827-1836.  
19745807 Y.Toh, D.Takeshita, T.Numata, S.Fukai, O.Nureki, and K.Tomita (2009).
Mechanism for the definition of elongation and termination by the class II CCA-adding enzyme.
  EMBO J, 28, 3353-3365.
PDB codes: 3h37 3h38 3h39 3h3a
18682528 A.Just, F.Butter, M.Trenkmann, T.Heitkam, M.Mörl, and H.Betat (2008).
A comparative analysis of two conserved motifs in bacterial poly(A) polymerase and CCA-adding enzyme.
  Nucleic Acids Res, 36, 5212-5220.  
18523015 A.Neuenfeldt, A.Just, H.Betat, and M.Mörl (2008).
Evolution of tRNA nucleotidyltransferases: a small deletion generated CC-adding enzymes.
  Proc Natl Acad Sci U S A, 105, 7953-7958.  
18177750 G.Martin, S.Doublié, and W.Keller (2008).
Determinants of substrate specificity in RNA-dependent nucleotidyl transferases.
  Biochim Biophys Acta, 1779, 206-216.  
18466919 M.Dupasquier, S.Kim, K.Halkidis, H.Gamper, and Y.M.Hou (2008).
tRNA integrity is a prerequisite for rapid CCA addition: implication for quality control.
  J Mol Biol, 379, 579-588.  
18302315 X.Shan, T.A.Russell, S.M.Paul, D.B.Kushner, and P.B.Joyce (2008).
Characterization of a temperature-sensitive mutation that impairs the function of yeast tRNA nucleotidyltransferase.
  Yeast, 25, 219-233.  
18583961 Y.Toh, T.Numata, K.Watanabe, D.Takeshita, O.Nureki, and K.Tomita (2008).
Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme.
  EMBO J, 27, 1944-1952.
PDB codes: 2zh1 2zh2 2zh3 2zh4 2zh5 2zh6 2zh7 2zh8 2zh9 2zha 2zhb
17872511 G.Martin, and W.Keller (2007).
RNA-specific ribonucleotidyl transferases.
  RNA, 13, 1834-1849.  
17179213 H.D.Cho, C.L.Verlinde, and A.M.Weiner (2007).
Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases.
  Proc Natl Acad Sci U S A, 104, 54-59.  
17189640 J.Stagno, I.Aphasizheva, A.Rosengarth, H.Luecke, and R.Aphasizhev (2007).
UTP-bound and Apo structures of a minimal RNA uridylyltransferase.
  J Mol Biol, 366, 882-899.
PDB codes: 2ikf 2nom
17850751 P.B.Balbo, and A.Bohm (2007).
Mechanism of poly(A) polymerase: structure of the enzyme-MgATP-RNA ternary complex and kinetic analysis.
  Structure, 15, 1117-1131.
PDB code: 2q66
16751877 A.A.Tulub (2006).
Molecular dynamics DFT:B3LYP study of guanosinetriphosphate conversion into guanosinemonophosphate upon Mg2+ chelation of alpha and beta phosphate oxygens of the triphosphate tail.
  Phys Chem Chem Phys, 8, 2187-2192.  
16455665 H.D.Cho, Y.Chen, G.Varani, and A.M.Weiner (2006).
A model for C74 addition by CCA-adding enzymes: C74 addition, like C75 and A76 addition, does not involve tRNA translocation.
  J Biol Chem, 281, 9801-9811.  
16809540 H.Oshikane, K.Sheppard, S.Fukai, Y.Nakamura, R.Ishitani, T.Numata, R.L.Sherrer, L.Feng, E.Schmitt, M.Panvert, S.Blanquet, Y.Mechulam, D.Söll, and O.Nureki (2006).
Structural basis of RNA-dependent recruitment of glutamine to the genetic code.
  Science, 312, 1950-1954.
PDB code: 2d6f
17051158 K.Tomita, R.Ishitani, S.Fukai, and O.Nureki (2006).
Complete crystallographic analysis of the dynamics of CCA sequence addition.
  Nature, 443, 956-960.
PDB codes: 2dr5 2dr7 2dr8 2dr9 2dra 2drb 2dvi
16028221 C.Lehmann, S.Pullalarevu, W.Krajewski, M.A.Willis, A.Galkin, A.Howard, and O.Herzberg (2005).
Structure of HI0073 from Haemophilus influenzae, the nucleotide-binding domain of a two-protein nucleotidyl transferase.
  Proteins, 60, 807-811.
PDB code: 1no5
16167380 D.Temiakov, N.Zenkin, M.N.Vassylyeva, A.Perederina, T.H.Tahirov, E.Kashkina, M.Savkina, S.Zorov, V.Nikiforov, N.Igarashi, N.Matsugaki, S.Wakatsuki, K.Severinov, and D.G.Vassylyev (2005).
Structural basis of transcription inhibition by antibiotic streptolydigin.
  Mol Cell, 19, 655-666.
PDB code: 2a6h
15590678 H.D.Cho, C.L.Verlinde, and A.M.Weiner (2005).
Archaeal CCA-adding enzymes: central role of a highly conserved beta-turn motif in RNA polymerization without translocation.
  J Biol Chem, 280, 9555-9566.  
16281058 J.Deng, N.L.Ernst, S.Turley, K.D.Stuart, and W.G.Hol (2005).
Structural basis for UTP specificity of RNA editing TUTases from Trypanosoma brucei.
  EMBO J, 24, 4007-4017.
PDB codes: 2b4v 2b51 2b56
15498478 A.M.Weiner (2004).
tRNA maturation: RNA polymerization without a nucleic acid template.
  Curr Biol, 14, R883-R885.  
15332079 P.Schimmel, and X.L.Yang (2004).
Two classes give lessons about CCA.
  Nat Struct Mol Biol, 11, 807-808.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.