PDBsum entry 3a2k

Ligase/RNA

PDB id: 3a2k

Contents

Protein chains

462 a.a. *

DNA/RNA

* Residue conservation analysis

PDB id:

3a2k

Name:

Ligase/RNA

Title:

Crystal structure of tils complexed with tRNA

Structure:

tRNA(ile)-lysidine synthase. Chain: a, b. Synonym: tRNA(ile)-lysidine synthetase, tRNA(ile)-2-lysyl-cytidine synthase. Engineered: yes. Bacterial tRNA. Chain: c, d. Engineered: yes

Source:

Geobacillus kaustophilus. Organism_taxid: 1462. Strain: hta426. Gene: gk0060, tils. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Other_details: RNA was prepared by in vitro transcript

Resolution:

3.65Å R-factor: 0.218 R-free: 0.266

Authors:

K.Nakanishi,L.Bonnefond,R.Ishitani,O.Nureki

Key ref:

K.Nakanishi et al. (2009). Structural basis for translational fidelity ensured by transfer RNA lysidine synthetase. Nature, 461, 1144-1148. PubMed id: 19847269 DOI: 10.1038/nature08474

Date:

23-May-09 Release date: 20-Oct-09

Protein chains

Q5L3T3  (TILS_GEOKA) -  tRNA(Ile)-lysidine synthase from Geobacillus kaustophilus (strain HTA426)

Seq: 464 a.a.

Struc: 462 a.a.

DNA/RNA chains

G-G-A-C-C-U-U-U-A-G-C-U-C-A-G-U-U-G-G-U-U-A-G-A-G-C-A-G-A-C-G-G-C-U-C-A-U-A-A- 77 bases

G-G-A-C-C-U-U-U-A-G-C-U-C-A-G-U-U-G-G-U-U-A-G-A-G-C-A-G-A-C-G-G-C-U-C-A-U-A-A- 77 bases

Enzyme reactions

• Enzyme class: E.C.6.3.4.19 - tRNA(Ile)-lysidine synthetase.
• Reaction: cytidine₃₄ in tRNA(Ile2) + L-lysine + ATP = lysidine₃₄ in tRNA(Ile2) + AMP + diphosphate + H⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1038/nature08474

Nature 461:1144-1148 (2009)

PubMed id: 19847269

Structural basis for translational fidelity ensured by transfer RNA lysidine synthetase.

K.Nakanishi, L.Bonnefond, S.Kimura, T.Suzuki, R.Ishitani, O.Nureki.

ABSTRACT

Maturation of precursor transfer RNA (pre-tRNA) includes excision of the 5' leader and 3' trailer sequences, removal of introns and addition of the CCA terminus. Nucleotide modifications are incorporated at different stages of tRNA processing, after the RNA molecule adopts the proper conformation. In bacteria, tRNA(Ile2) lysidine synthetase (TilS) modifies cytidine into lysidine (L; 2-lysyl-cytidine) at the first anticodon of tRNA(Ile2) (refs 4-9). This modification switches tRNA(Ile2) from a methionine-specific to an isoleucine-specific tRNA. However, the aminoacylation of tRNA(Ile2) by methionyl-tRNA synthetase (MetRS), before the modification by TilS, might lead to the misincorporation of methionine in response to isoleucine codons. The mechanism used by bacteria to avoid this pitfall is unknown. Here we show that the TilS enzyme specifically recognizes and modifies tRNA(Ile2) in its precursor form, thereby avoiding translation errors. We identified the lysidine modification in pre-tRNA(Ile2) isolated from RNase-E-deficient Escherichia coli and did not detect mature tRNA(Ile2) lacking this modification. Our kinetic analyses revealed that TilS can modify both types of RNA molecule with comparable efficiencies. X-ray crystallography and mutational analyses revealed that TilS specifically recognizes the entire L-shape structure in pre-tRNA(Ile2) through extensive interactions coupled with sequential domain movements. Our results demonstrate how TilS prevents the recognition of tRNA(Ile2) by MetRS and achieves high specificity for its substrate. These two key points form the basis for maintaining the fidelity of isoleucine codon translation in bacteria. Our findings also provide a rationale for the necessity of incorporating specific modifications at the precursor level during tRNA biogenesis.

Selected figure(s)

Figure 2.
Figure 2: tRNA recognition by GkTilS. a, Overall homodimeric structure. b, Recognition of the anticodon loop. F[o ]- F[c] simulated annealing omit maps (3.7 ) of Arg 142 and three nucleotides (C32, C34 and A38) are shown in magenta and grey, respectively. c, Left, recognition of the major groove of the acceptor stem by the HTH motif and -hairpin; right, the protein surface that complementarily interacts with the 3'-ACCA terminus is coloured according to its electrostatic potential. d, Lysidine incorporating activities of tRNA^Ile2 and GkTilS mutants. The initial rate of lysidine incorporation is shown. Error bars, s.d. of three independent experiments.

Figure 3.
Figure 3: Sequential tRNA recognition mechanism. a, Superimposition of apo-EcTilS and tRNA-bound GkTilS on their catalytic domains. b, Superimposition of type I and II TilSs on their SCL domains. c, Lysidine incorporation into tRNA^Mets by GkTilS (left) and AaTilS (right). Error bars denote s.d. of three independent experiments. d, Lysidine formation model. e, Apo-form (apo-EcTilS): the ASB and SCL domains have an intramolecular hydrophobic interaction. f, Initial binding state (manual docking of yeast tRNA^Phe (PDB accession 1EHZ) onto the apo-EcTilS): capture of the pre-tRNA^Ile2 acceptor stem by the ASB domain triggers disruption of the hydrophobic interactions. g, Pre-reaction state (the current structure): drastic domain movements allow TilS to interact fully with tRNA.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2009, 461, 1144-1148) copyright 2009.

Figures were selected by an automated process.