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Identification of the active site of poly(A)-specific ribonuclease by site-directed mutagenesis and Fe(2+)-mediated

Yan-Guo Ren1, Javier Martínez, Anders Virtanen

  • 1Department of Cell and Molecular Biology, Uppsala University, BMC, Box 596, SE-751 24 Uppsala, Sweden.

The Journal of Biological Chemistry
|December 14, 2001
PubMed

Insights

Poly(A)-specific ribonuclease (PARN) uses four key acidic amino acids in its active site for catalysis. These residues are crucial for binding metal ions, similar to E. coli DNA polymerase I.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Poly(A)-specific ribonuclease (PARN) is a unique mammalian exoribonuclease.
  • It specifically degrades the mRNA poly(A) tail.
  • PARN belongs to the RNase D family, defined by conserved acidic residues.

Purpose of the Study:

  • To investigate the role of conserved acidic residues in human PARN function.
  • To identify metal ion binding sites within the PARN active site.
  • To compare the PARN active site structure with other nucleases.

Main Methods:

  • Site-directed mutagenesis was used to alter conserved acidic residues.
  • Iron(II)-induced hydroxyl radical cleavage mapped Fe(2+) binding sites.
  • Apparent dissociation constants ((app)K(d)) were measured for Fe(2+) binding.

Main Results:

  • Mutating Asp(28), Glu(30), Asp(292), and Asp(382) showed they are essential for catalysis.
  • These residues are not required for PARN-RNA substrate complex stabilization.
  • Three conserved acidic residues were important for Fe(2+) binding at two identified sites.
  • Mutations affecting these residues altered Fe(2+) binding affinity, indicating metal ion coordination.

Conclusions:

  • The four conserved acidic amino acids are essential active site residues in PARN.
  • These residues coordinate divalent metal ions.
  • The PARN active site shares functional and structural similarities with the 3'-exonuclease domain of E. coli DNA polymerase I.

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