PDBsum entry: 1q78

Transferase

PDB id: 1q78

Contents

Protein chain

470 a.a. *

Ligands

3AT

Metals

_MG

Waters ×123

* Residue conservation analysis

PDB id:

1q78

Name:

Transferase

Title:

Crystal structure of poly(a) polymerase in complex with 3'- datp and magnesium chloride

Structure:

Poly(a) polymerase alpha. Chain: a. Fragment: residues 1-513. Synonym: pap, polynucleotide adenylyltransferase alpha. Engineered: yes

Source:

Bos taurus. Cattle. Organism_taxid: 9913. Gene: papola or pap. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.80Å R-factor: 0.204 R-free: 0.255

Authors:

G.Martin,A.Moglich,W.Keller,S.Doublie

Key ref:

G.Martin et al. (2004). Biochemical and structural insights into substrate binding and catalytic mechanism of mammalian poly(A) polymerase. J Mol Biol, 341, 911-925. PubMed id: 15328606 DOI: 10.1016/j.jmb.2004.06.047

Date:

16-Aug-03 Release date: 07-Sep-04

Protein chain

P25500  (PAPOA_BOVIN) -  Poly(A) polymerase alpha

Seq: 739 a.a.

Struc: 470 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.7.7.19 - Polynucleotide adenylyltransferase.
• Reaction: ATP + RNA(n) = diphosphate + RNA(n+1)

ATP (Bound ligand (Het Group name = 3AT) matches with 96.00% similarity) + RNA(n) = diphosphate + RNA(n+1)

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

 Gene Ontology (GO) functional annotation 

• Cellular component: nucleus (1 term)
• Biological process: transcription (3 terms)
• Biochemical function: RNA binding (3 terms)

reference

DOI no: 10.1016/j.jmb.2004.06.047

J Mol Biol 341:911-925 (2004)

PubMed id: 15328606

Biochemical and structural insights into substrate binding and catalytic mechanism of mammalian poly(A) polymerase.

G.Martin, A.Möglich, W.Keller, S.Doublié.

ABSTRACT

Polyadenylation of messenger RNA precursors is an essential process in eukaryotes. Poly(A) polymerase (PAP), a member of the nucleotidyltransferase family that includes DNA polymerase beta, incorporates ATP at the 3' end of mRNAs in a template-independent manner. Although the structures of mammalian and yeast PAPs are known, their mechanism of ATP selection has remained elusive. In a recent bovine PAP structure complexed with an analog of ATP and Mn2+, strictly conserved residues interact selectively with the adenine base, but the nucleotide was found in a "non-productive" conformation. Here we report a second bovine crystal structure, obtained in the presence of Mg2+, where 3'-dATP adopts a "productive" conformation similar to that seen in yeast PAP or DNA polymerase beta. Mutational analysis and activity assays with ATP analogs suggest a role in catalysis for one of the two adenine-binding sites revealed by our structural data. The other site might function to prevent futile hydrolysis of ATP. In order to investigate the role of metals in catalysis we performed steady state kinetics experiments under distributive polymerization conditions. These tests suggest a sequential random mechanism in vitro in the presence of ATP and RNA, without preference for a particular order of binding of the two substrates. In vivo, however, where polyadenylation is processive and the primer does not dissociate from the enzyme, an ordered mechanism with the primer as the leading substrate is more likely.

Selected figure(s)

Figure 2.
Figure 2. A, Cartoon of the interactions between PAP and 3'-dATP in site A. B, Cartoon of the interactions between PAP and 3'-dATP in site B. For clarity, only the interactions with the base and ribose are shown. Hydrophobic interactions are shown as spoked arcs, and hydrogen bonds as dotted lines.57

Figure 4.
Figure 4. Comparison of nucleotide conformation in Mg2+ (blue) and Mn2+-bound (pink) PAP structures superimposed with Pol b structure (grey; PDB No. 1BPY) including DNA primer and metals. The a-phosphates are indicated by circles (Mn2+-bound: hot pink; Mg2+-bound: dark blue; and Pol b: dark grey) and the modeled 3'-OH of the Pol b primer (3' deoxy in the crystal) by a grey sphere. The distances between the nucleophile and a-phosphate groups are shown as dotted lines. The proposed primer-binding motif is highlighted in yellow.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2004, 341, 911-925) copyright 2004.

Figures were selected by an automated process.