The activity and selectivity of fission yeast Pop2p are affected by a high affinity for Zn2+ and Mn2+ in the active site

TY - JOUR

T1 - The activity and selectivity of fission yeast Pop2p are affected by a high affinity for Zn2+ and Mn2+ in the active site

AU - Andersen, Kasper Røjkjær

AU - Jonstrup, Anette Thyssen

AU - Van, Lan Bich

AU - Brodersen, Ditlev Egeskov

PY - 2009/5

Y1 - 2009/5

N2 - In eukaryotic organisms, initiation of mRNA turnover is controlled by progressive shortening of the poly-A tail, a process involving the mega-Dalton Ccr4-Not complex and its two associated 3'-5' exonucleases, Ccr4p and Pop2p (Caf1p). RNA degradation by the 3'-5' DEDDh exonuclease, Pop2p, is governed by the classical two metal ion mechanism traditionally assumed to be dependent on Mg(2+) ions bound in the active site. Here, we show biochemically and structurally that fission yeast (Schizosaccharomyces pombe) Pop2p prefers Mn(2+) and Zn(2+) over Mg(2+) at the concentrations of the ions found inside cells and that the identity of the ions in the active site affects the activity of the enzyme. Ion replacement experiments further suggest that mRNA deadenylation could be subtly regulated by local Zn(2+) levels in the cell. Finally, we use site-directed mutagenesis to propose a mechanistic model for the basis of the preference for poly-A sequences exhibited by the Pop2p-type deadenylases as well as their distributive enzymatic behavior.

AB - In eukaryotic organisms, initiation of mRNA turnover is controlled by progressive shortening of the poly-A tail, a process involving the mega-Dalton Ccr4-Not complex and its two associated 3'-5' exonucleases, Ccr4p and Pop2p (Caf1p). RNA degradation by the 3'-5' DEDDh exonuclease, Pop2p, is governed by the classical two metal ion mechanism traditionally assumed to be dependent on Mg(2+) ions bound in the active site. Here, we show biochemically and structurally that fission yeast (Schizosaccharomyces pombe) Pop2p prefers Mn(2+) and Zn(2+) over Mg(2+) at the concentrations of the ions found inside cells and that the identity of the ions in the active site affects the activity of the enzyme. Ion replacement experiments further suggest that mRNA deadenylation could be subtly regulated by local Zn(2+) levels in the cell. Finally, we use site-directed mutagenesis to propose a mechanistic model for the basis of the preference for poly-A sequences exhibited by the Pop2p-type deadenylases as well as their distributive enzymatic behavior.

KW - Amino Acid Sequence

KW - Catalytic Domain

KW - Crystallography, X-Ray

KW - Manganese

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Poly A

KW - Ribonucleases

KW - Schizosaccharomyces

KW - Schizosaccharomyces pombe Proteins

KW - Sequence Alignment

KW - Zinc

U2 - 10.1261/rna.1489409

DO - 10.1261/rna.1489409

M3 - Journal article

C2 - 19307292

SN - 1355-8382

VL - 15

SP - 850

EP - 861

JO - RNA

JF - RNA

IS - 5

ER -