PDBsum entry 1vfg

Transferase/RNA

PDB id: 1vfg

Contents

Protein chains

342 a.a. *

DNA/RNA

Ligands

APC ×2

Waters ×79

* Residue conservation analysis

PDB id:

1vfg

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)

Resolution:

2.80Å R-factor: 0.230 R-free: 0.287

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

Date:

13-Apr-04 Release date: 10-Aug-04

Protein chains

O66728  (O66728_AQUAE) -  A-adding tRNA nucleotidyltransferase from Aquifex aeolicus (strain VF5)

Seq: 824 a.a.

Struc: 342 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DNA/RNA chains

G-G-C-C-A-G-G-G-G-C-G-G-U-U-C-G-A-U-U-C-C-G-C-C-C-C-U-G-G-C-C 31 bases

G-G-C-C-A-G-G-G-G-C-G-G-U-U-C-G-A-U-U-C-C-G-C-C-C-C-U-G-G-C-C-A-C-C 34 bases

Enzyme reactions

• Enzyme class: E.C.2.7.7.- - ?????

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.

ABSTRACT

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.