Polynucleotide adenylyltransferase

 

Poly(A) polymerase (PAP) catalyses the addition of a poly-adenosine tail to almost all eukaryotic mRNAs. This poly(A) tail has numerous functions in eukaryotes: it facilitates transport of the mRNA from the nucleus, regulates mRNA stability, and increases the efficiency of translation. Poly(A) tails are added to mRNA by a multiprotein complex that recognises the polyadenylation signal, cleaves the precursor mRNA, and adds the additional nucleotides. The poly(A) polymerase component retains its polymerase activity when isolated from the holoenzyme assembly and will progressively add long stretches of adenosine nucleotides to an RNA primer in vitro. Across eukaryotes, it appears the catalytic part of the enzyme resides in the NTD while regulation occurs in the CTD.

 

Reference Protein and Structure

Sequence
P29468 UniProt (2.7.7.19) IPR014492 (Sequence Homologues) (PDB Homologues)
Biological species
Saccharomyces cerevisiae S288c (Baker's yeast) Uniprot
PDB
1fa0 - STRUCTURE OF YEAST POLY(A) POLYMERASE BOUND TO MANGANATE AND 3'-DATP (2.6 Å) PDBe PDBsum 1fa0
Catalytic CATH Domains
1.10.1410.10 CATHdb 3.30.460.10 CATHdb (see all for 1fa0)
Cofactors
Magnesium(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:2.7.7.19)

ATP(4-)
CHEBI:30616ChEBI
+
RNA (poly(A))
CHEBI:8756ChEBI
→
diphosphate(3-)
CHEBI:33019ChEBI
+
RNA (poly(A))
CHEBI:8756ChEBI
Alternative enzyme names: AMP polynucleotidylexotransferase, ATP-polynucleotide adenylyltransferase, ATP:polynucleotidylexotransferase, NTP polymerase, RNA adenylating enzyme, RNA formation factors, PF1, Adenosine triphosphate:ribonucleic acid adenylyltransferase, Poly(A) hydrolase, Poly(A) polymerase, Poly(A) synthetase, Polyadenylate nucleotidyltransferase, Polyadenylate polymerase, Polyadenylate synthetase, Polyadenylic acid polymerase, Polyadenylic polymerase, Terminal riboadenylate transferase,

Enzyme Mechanism

Introduction

From it's crystal structures, Poly(A) polymerase is thought to use the two-metal-ion mechanism proposed for all nucleic acid polymerases. One magnesium ion contacts the 3' OH of the primer and lowers its pKa, activating it for in-line nucleophilic attack on the alpha phosphate of the incoming ATP. Accumulation of negative charge on the alpha phosphate in the pentacoordinate transition state is stabilised by both magnesium ions. The second magnesium ion additionally functions to stabilise accumulation of negative charge on the pyrophosphate leaving group alongside Lys 215.

Catalytic Residues Roles

UniProt PDB* (1fa0)
Asp154 Asp154A Coordinates to metal ion A, a magnesium cation which activates the 3' OH on the ribose sugar for nucleophilic attack. metal ligand
Asp102, Asp100 Asp102A, Asp100A Coordinates to metal ion B, a magnesium cation which in turn stabilises the negative charges forming in the transition state of the reaction. metal ligand
Tyr224, Ser89, Lys215 Tyr224A, Ser89A, Lys215A Form hydrogen bonds and salt bridge interactions with ATP's triphosphate group, helping stabilise the negatively charged transition state that forms in the reaction. electrostatic stabiliser, polar interaction
*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

bimolecular nucleophilic substitution, inferred reaction step, overall product formed, overall reactant used

References

  1. Bard J et al. (2000), Science, 289, 1346-1349. Structure of Yeast Poly(A) Polymerase Alone and in Complex with 3'-dATP. DOI:10.1126/science.289.5483.1346. PMID:10958780.
  2. Yang Q et al. (2014), J Mol Biol, 426, 43-50. Crystal structure of human poly(A) polymerase gamma reveals a conserved catalytic core for canonical poly(A) polymerases. DOI:10.1016/j.jmb.2013.09.025. PMID:24076191.
  3. Balbo PB et al. (2007), Structure, 15, 1117-1131. Mechanism of poly(A) polymerase: structure of the enzyme-MgATP-RNA ternary complex and kinetic analysis. DOI:10.1016/j.str.2007.07.010. PMID:17850751.
  4. Brautigam CA et al. (1998), Curr Opin Struct Biol, 8, 54-63. Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes. DOI:10.1016/s0959-440x(98)80010-9. PMID:9519297.
  5. Doublié S et al. (1998), Curr Opin Struct Biol, 8, 704-712. The mechanism of action of T7 DNA polymerase. DOI:10.1016/s0959-440x(98)80089-4. PMID:9914251.

Step 1. Mg2+ coordinating to the 3' OH on the ribose sugar lowers the pka of the surrounding area, increasing it's nucleophilicity to attack the alpha phosphate on ATP.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys215A electrostatic stabiliser
Tyr224A electrostatic stabiliser
Ser89A electrostatic stabiliser
Asp100A metal ligand
Asp102A metal ligand
Asp154A metal ligand
Ser89A polar interaction
Lys215A polar interaction
Tyr224A polar interaction

Chemical Components

ingold: bimolecular nucleophilic substitution, inferred reaction step, overall product formed, overall reactant used
Download: Image, Marvin File

Step 1. Mg2+ coordinating to the 3' OH on the ribose sugar lowers the pka of the surrounding area, increasing it's nucleophilicity to attack the alpha phosphate on ATP.

Products.

Contributors

Steven Smith, Gemma L. Holliday, Morwenna Hall