RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2

J Biol Chem. 2004 Jul 23;279(30):31337-47. doi: 10.1074/jbc.M402394200. Epub 2004 Apr 13.

Abstract

Here we report that bacteriophage T4 RNA ligase 2 (Rnl2) is an efficient catalyst of RNA ligation at a 3'-OH/5'-PO(4) nick in a double-stranded RNA or an RNA.DNA hybrid. The critical role of the template strand in approximating the reactive 3'-OH and 5'-PO(4) termini is underscored by the drastic reductions in the RNA-sealing activity of Rnl2 when the duplex substrates contain gaps or flaps instead of nicks. RNA nick joining requires ATP and a divalent cation cofactor (either Mg or Mn). Neither dATP, GTP, CTP, nor UTP can substitute for ATP. We identify by alanine scanning seven functionally important amino acids (Tyr-5, Arg-33, Lys-54, Gln-106, Asp-135, Arg-155, and Ser-170) within the N-terminal nucleotidyl-transferase domain of Rnl2 and impute specific roles for these residues based on the crystal structure of the AMP-bound enzyme. Mutational analysis of 14 conserved residues in the C-terminal domain of Rnl2 identifies 3 amino acids (Arg-266, Asp-292, and Glu-296) as essential for ligase activity. Our findings consolidate the evolutionary connections between bacteriophage Rnl2 and the RNA-editing ligases of kinetoplastid protozoa.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacteriophage T4 / enzymology*
  • Bacteriophage T4 / genetics*
  • Base Sequence
  • DNA, Viral / genetics
  • DNA, Viral / metabolism
  • Genes, Viral
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis
  • Protein Structure, Tertiary
  • RNA Ligase (ATP) / chemistry
  • RNA Ligase (ATP) / genetics*
  • RNA Ligase (ATP) / metabolism*
  • RNA, Fungal / genetics
  • RNA, Fungal / metabolism
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Viral Proteins / chemistry
  • Viral Proteins / genetics*
  • Viral Proteins / metabolism*

Substances

  • DNA, Viral
  • RNA, Fungal
  • Viral Proteins
  • RNA Ligase (ATP)
  • bacteriophage T4 RNA ligase 2