Polynucleotide 5'-phosphatase

 

RNA triphosphatase is an essential mRNA processing enzyme that catalyses the first step in cap formation. Capping entails three enzymatic reactions in which the 5' triphosphate end of pre-mRNA is hydrolysed to a 5' diphosphate by RNA triphosphatase, then capped with GMP by RNA guanylyltransferase, and methylated by RNA methyltransferase. Each of the capping activities is essential for cell growth.

Yeast RNA triphosphatase forms a heteromeric complex with the yeast RNA guanylyltransferase . The binding serves two purposes, firstly, the interaction stimulates guanylyltransferase activity by enhancing the affinity of RNA guanylyltransferase for GTP. Secondly the tethering facilitates the recruitment of the triphosphatase to the RNA polymerase II elongation complex.

The structure reveals a novel active site fold, a topologically closed trisphosphate tunnel formed from an eight stranded beta barrel.

 

Reference Protein and Structure

Sequence
O13297 UniProt (3.1.3.33) IPR004206 (Sequence Homologues) (PDB Homologues)
Biological species
Saccharomyces cerevisiae S288c (Baker's yeast) Uniprot
PDB
1d8h - X-RAY CRYSTAL STRUCTURE OF YEAST RNA TRIPHOSPHATASE IN COMPLEX WITH SULFATE AND MANGANESE IONS. (2.0 Å) PDBe PDBsum 1d8h
Catalytic CATH Domains
3.20.100.10 CATHdb (see all for 1d8h)
Cofactors
Manganese(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.1.3.33)

water
CHEBI:15377ChEBI
+
5'-phosphopolynucleotide
CHEBI:1981ChEBI
→
polynucleotide
CHEBI:15986ChEBI
+
phosphoric acid
CHEBI:26078ChEBI
Alternative enzyme names: 5'-polynucleotidase, Polynucleotide 5'-triphosphatase,

Enzyme Mechanism

Introduction

The 5'-phosphopolynucleotide substrate is hydrolysed into its polynucleotide and phosphate components. Structural data suggests a mechanism whereby acid residues located on the floor of the tunnel coordinate a catalytically essential divalent cation that in turn coordinates the gamma-phosphate. The metal ion activates the gamma-phosphate for attack by water and stabilises a pentavalent phosphorous transition state in which the attacking water is apical to the beta-phosphate leaving group. The basic side chains of Arg393, Lys409 and Lys456 stabilise the developing negative charge on the gamma-phosphate in the transition state.

Catalytic Residues Roles

UniProt PDB* (1d8h)
Lys409 Lys409(171)A The basic side chain stabilises the developing negative charge on the gamma-phosphate in the transition state. electrostatic stabiliser
Arg393, Lys456 Arg393(155)A, Lys456(218)A The basic side chain stabilises the developing negative charge on the gamma-phosphate in the transition state. electrostatic stabiliser
Glu305, Glu496, Glu307 Glu305(67)A, Glu496(258)A, Glu307(69)A Forms the Mn(II) binding site. metal ligand
*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

References

  1. Massayuki Kikuti C et al. (2006), Mol Biochem Parasitol, 150, 83-95. Divalent metal requirements for catalysis and stability of the RNA triphosphatase from Trypanosoma cruzi. DOI:10.1016/j.molbiopara.2006.06.012. PMID:16887207.
  2. Lima CD et al. (1999), Cell, 99, 533-543. Structure and mechanism of yeast RNA triphosphatase: an essential component of the mRNA capping apparatus. PMID:10589681.

Step 1.

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Catalytic Residues Roles

Residue Roles
Glu305(67)A metal ligand
Glu307(69)A metal ligand
Glu496(258)A metal ligand
Lys456(218)A electrostatic stabiliser
Arg393(155)A electrostatic stabiliser
Lys409(171)A electrostatic stabiliser

Chemical Components

Step 1.

Contributors

James W. Murray, Craig Porter, Gemma L. Holliday