PDBsum entry 2q66

Transferase/RNA

PDB id: 2q66

Contents

Protein chain

519 a.a. *

DNA/RNA

Ligands

EDO ×17

ATP

Metals

_MG

Waters ×509

* Residue conservation analysis

PDB id:

2q66

Name:

Transferase/RNA

Title:

Structure of yeast poly(a) polymerase with atp and oligo(a)

Structure:

5'-r(p Ap Ap Ap Ap A)-3'. Chain: x. Engineered: yes. Poly(a) polymerase. Chain: a. Synonym: pap, polynucleotide adenylyltransferase. Engineered: yes. Mutation: yes

Source:

Synthetic: yes. Other_details: synthetic. Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Gene: pap1. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.80Å R-factor: 0.191 R-free: 0.225

Authors:

A.Bohm,P.Balbo

Key ref:

P.B.Balbo and A.Bohm (2007). Mechanism of Poly(A) Polymerase: Structure of the Enzyme-MgATP-RNA Ternary Complex and Kinetic Analysis. Structure, 15, 1117-1131. PubMed id: 17850751 DOI: 10.1016/j.str.2007.07.010

Date:

04-Jun-07 Release date: 28-Aug-07

Protein chain

P29468  (PAP_YEAST) -  Poly(A) polymerase from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 568 a.a.

Struc: 519 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DNA/RNA chain

A-A-A-A-A 5 bases

Enzyme reactions

• Enzyme class: E.C.2.7.7.19 - polynucleotide adenylyltransferase.
• Reaction: RNA(n) + ATP = RNA(n)-3'-adenine ribonucleotide + diphosphate

RNA(n) (Bound ligand (Het Group name = ATP) corresponds exactly) + ATP = RNA(n)-3'-adenine ribonucleotide + diphosphate

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

reference

DOI no: 10.1016/j.str.2007.07.010

Structure 15:1117-1131 (2007)

PubMed id: 17850751

Mechanism of Poly(A) Polymerase: Structure of the Enzyme-MgATP-RNA Ternary Complex and Kinetic Analysis.

P.B.Balbo, A.Bohm.

ABSTRACT

We report the 1.8 A structure of yeast poly(A) polymerase (PAP) trapped in complex with ATP and a five residue poly(A) by mutation of the catalytically required aspartic acid 154 to alanine. The enzyme has undergone significant domain movement and reveals a closed conformation with extensive interactions between the substrates and all three polymerase domains. Both substrates and 31 buried water molecules are enclosed within a central cavity that is open at both ends. Four PAP mutants were subjected to detailed kinetic analysis, and studies of the adenylyltransfer (forward), pyrophosphorolysis (reverse), and nucleotidyltransfer reaction utilizing CTP for the mutants are presented. The results support a model in which binding of both poly(A) and the correct nucleotide, MgATP, induces a conformational change, resulting in formation of a stable, closed enzyme state. Thermodynamic considerations of the data are discussed as they pertain to domain closure, substrate specificity, and catalytic strategies utilized by PAP.

Selected figure(s)

Figure 2.
Figure 2. Determinants of ATP and RNA Binding
Interacting residues from the N-terminal, middle, and C-terminal domains are colored yellow, green, and cyan, respectively. Residues within cloud-shaped bubbles on either side of the bases indicate hydrophobic/van der Waals interactions. Water molecules (circles) are color coded based on the degree to which they are buried within the interior of the protein; those colored dark blue are completely buried (see Experimental Procedures). Those colored gray-blue are at the protein surface. Others are colored intermediate shades of blue depending on how many shells of water needed to be removed in order for the water atoms to become exposed. Asp154 and the second Mg^2+ ion are shown in gray. Eight additional water molecules interact with the triphosphate moiety of the ATP. These are not shown for clarity, and because the water structure in this region may be altered due to the D154A mutation. The base at position −5 interacts with a neighboring PAP molecule and is not shown.

Figure 3.
Figure 3. Stereo Views of the Substrate Binding Sites of PAP
Detailed substrate interactions formed in the closed, ternary complex. ATP (yellow carbons) and the 3′ end (blue carbons) are shown along with PAP with the surrounding amino acids (green carbons) and water molecules (red spheres).

The above figures are reprinted from an Open Access publication published by Cell Press: Structure (2007, 15, 1117-1131) copyright 2007.

Figures were selected by an automated process.