TRNA-intron lyase

 

RNA endonuclease from the archaea Methanocaldococcus jannaschii is able to catalyse the splicing of introns from tRNA, a process needed only in archaea and eukarya, bacterial tRNA introns being self-splicing. The enzyme thus is a homologue of the eukaryotic enzymes which perform, a fact that serves to reinforce the closer relationship proposed between archaea and eukarya. There are differences however in the exact orientation of residues at the active site and the substrate recognition by the enzymes, suggesting that the enzymes evolved from a common ancestor with slightly different function.

 

Reference Protein and Structure

Sequence
Q58819 UniProt (4.6.1.16) IPR016442 (Sequence Homologues) (PDB Homologues)
Biological species
Methanocaldococcus jannaschii DSM 2661 (Archaea) Uniprot
PDB
1a79 - CRYSTAL STRUCTURE OF THE TRNA SPLICING ENDONUCLEASE FROM METHANOCOCCUS JANNASCHII (2.28 Å) PDBe PDBsum 1a79
Catalytic CATH Domains
3.40.1350.10 CATHdb (see all for 1a79)
Click To Show Structure

Enzyme Reaction (EC:4.6.1.16)

transfer RNA
CHEBI:17843ChEBI
+
water
CHEBI:15377ChEBI
→
hydron
CHEBI:15378ChEBI
+
5'-phosphate ribonucleotide-(2'-deoxyribonucleotide) residue(3-)
CHEBI:136415ChEBI
+
nucleoside 5'-monophosphate(2-)
CHEBI:58043ChEBI
Alternative enzyme names: Splicing endonuclease, tRNA splicing endonuclease, tRNATRPintron endonuclease, Transfer ribonucleate intron endoribonuclease, Transfer splicing endonuclease,

Enzyme Mechanism

Introduction

The overall reaction catalysed by the endonuclease is the formation of a 5'-OH and a 2',3'-cyclic phosphate, thus cleaving the RNA. This occurs through nucleophilic attack by the 2'-OH on the phosphate facilitated by deprotonation by His 125, which dissociates from the 5' via protonation by Tyr 115 via a pentavalent phosphate transition state stabilised by Lys 156. Subsequent hydrolysis of the produced 2',3'-cyclic phosphate completes the reaction.

Catalytic Residues Roles

UniProt PDB* (1a79)
Tyr115 Tyr115(107)A Protonates the 5' Oxygen to allow it to act as a leaving group in the reaction thus facilitating an SN2 displacement at the electrophilic phosphorous centre. increase nucleophilicity, promote heterolysis, proton acceptor, proton donor
His125 His125(117)A Deprotonates the 2'OH group to allow it to act as a nucleophile and attack the electrophilic phosphorous forming the pentavalent phosphate transition state. increase nucleophilicity, promote heterolysis, proton acceptor, proton donor
Lys156 Lys156(148)A Stabilises the pentavalent phosphate that forms in the reaction through electrostatic interactions. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

cyclisation, intramolecular nucleophilic substitution, proton transfer, overall product formed, overall reactant used, native state of enzyme regenerated, decyclisation, bimolecular nucleophilic substitution

References

  1. Li H et al. (1998), Science, 280, 279-284. Crystal Structure and Evolution of a Transfer RNA Splicing Enzyme. DOI:10.1126/science.280.5361.279. PMID:9535656.
  2. Calvin K et al. (2008), Biochemistry, 47, 13659-13665. Probing the catalytic triad of an archaeal RNA splicing endonuclease. DOI:10.1021/bi801141q. PMID:19053288.
  3. Kim YK et al. (2007), J Bacteriol, 189, 8339-8346. Structural and mutational analysis of tRNA intron-splicing endonuclease from Thermoplasma acidophilum DSM 1728: catalytic mechanism of tRNA intron-splicing endonucleases. DOI:10.1128/JB.00713-07. PMID:17827289.

Step 1. His125 deprotonates the 2' hydroxyl group this promotes its nucleophilic attack on the phosphate group forming a cyclic intermediate. Tyr115 protonates the the 5' hydroxyl leaving group promoting the cleavage. This SN2 reaction proceeds via a penta-coordinate transition state stabilized by Lys156.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys156(148)A electrostatic stabiliser
His125(117)A increase nucleophilicity
Tyr115(107)A promote heterolysis
His125(117)A proton acceptor
Tyr115(107)A proton donor

Chemical Components

cyclisation, ingold: intramolecular nucleophilic substitution, proton transfer, overall product formed, overall reactant used

Step 2. Tyr115 now acting as a base deprotonates a water molecule allowing it to perform a nucleophillic attack on the phosphate group. This leads to the cleavage of the 2' hydroxyl- phosphate bond upon protonation by His125.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys156(148)A electrostatic stabiliser
His125(117)A promote heterolysis
Tyr115(107)A increase nucleophilicity
Tyr115(107)A proton acceptor
His125(117)A proton donor

Chemical Components

proton transfer, native state of enzyme regenerated, overall product formed, decyclisation, ingold: bimolecular nucleophilic substitution
Download: Image, Marvin File

Step 1. His125 deprotonates the 2' hydroxyl group this promotes its nucleophilic attack on the phosphate group forming a cyclic intermediate. Tyr115 protonates the the 5' hydroxyl leaving group promoting the cleavage. This SN2 reaction proceeds via a penta-coordinate transition state stabilized by Lys156.

Step 2. Tyr115 now acting as a base deprotonates a water molecule allowing it to perform a nucleophillic attack on the phosphate group. This leads to the cleavage of the 2' hydroxyl- phosphate bond upon protonation by His125.

Products.

Contributors

Peter Sarkies, Gemma L. Holliday, James Willey