Identification of the active site of poly(A)-specific ribonuclease by site-directed mutagenesis and Fe(2+)-mediated cleavage.
Poly(A)-specific ribonuclease (PARN) is the only mammalian exoribonuclease characterized thus far with high specificity for degrading the mRNA poly(A) tail. PARN belongs to the RNase D family of nucleases, a family characterized by the presence of four conserved acidic amino acid residues. Here, we show by site-directed mutagenesis that these residues of human PARN, i.e. Asp(28), Glu(30), Asp(292), and Asp(382), are essential for catalysis but are not required for stabilization of the PARN x RNA substrate complex. We have used iron(II)-induced hydroxyl radical cleavage to map Fe(2+) binding sites in PARN. Two Fe(2+) binding sites were identified, and three of the conserved acidic amino acid residues were important for Fe(2+) binding at these sites. Furthermore, we show that the apparent dissociation constant ((app)K(d)) values for Fe(2+) binding at both sites were affected in PARN polypeptides in which the conserved acidic amino acid residues were substituted to alanine. This suggests that these residues coordinate divalent metal ions. We conclude that the four conserved acidic amino acids are essential residues of the PARN active site and that the active site of PARN functionally and structurally resembles the active site for 3'-exonuclease domain of Escherichia coli DNA polymerase I.
Ren
Yan-Guo
YG
Department of Cell and Molecular Biology, Uppsala University, BMC, Box 596, SE-751 24 Uppsala, Sweden.
Martínez
Javier
J
Virtanen
Anders
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid; D001665:Binding Sites; D005095:Exoribonucleases / Q000737:chemistry / Q000378:metabolism*; D018698:Glutamic Acid; D006801:Humans; D007501:Iron / Q000494:pharmacology*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001224:Aspartic Acid; D005095:Exoribonucleases; D018698:Glutamic Acid; D007501:Iron; D011994:Recombinant Proteins