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

  


Authors: Nandakumar, J.; Ho, C. K.; Lima, C. D.; Shuman, S.
Article Title: RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2
Abstract: Here we report that bacteriophage T4 RNA ligase 2 (Rnl2) is an efficient catalyst of RNA ligation at a 3′-OH/ 5′-PO4 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.
Keywords: controlled study; nonhuman; serine; carboxy terminal sequence; enzyme activity; tyrosine; mutational analysis; rna; dna; molecular evolution; amino acid sequence; molecular sequence data; sequence homology, amino acid; amino terminal sequence; substrate specificity; base sequence; dna, viral; amino acid; crystal structure; models, molecular; protein structure, tertiary; alanine; amino acids; adenosine triphosphate; nick end labeling; enzyme specificity; enzyme structure; glutamic acid; guanosine triphosphate; aspartic acid; mutagenesis; protozoa; genetic conservation; catalyst; enzymes; glutamine; lysine; rna ligase (atp); protozoon; arginine; double stranded rna; rna, fungal; viral proteins; divalent cation; adenosine phosphate; nucleotidyltransferase; magnesium; dna rna hybridization; bacteria; catalysts; manganese; rna ligase; bacteriophage; cytidine triphosphate; deoxyadenosine triphosphate; genes, viral; rna editing; bacteriophage t4; kinetoplastida; adenosinetriphosphate; uridine triphosphate; priority journal; article; kinetoplastid protozoa; rna substrate; unidentified bacteriophage
Journal Title: Journal of Biological Chemistry
Volume: 279
Issue: 30
ISSN: 0021-9258
Publisher: American Society for Biochemistry and Molecular Biology  
Date Published: 2004-07-23
Start Page: 31337
End Page: 31347
Language: English
DOI: 10.1074/jbc.M402394200
PROVIDER: scopus
PUBMED: 15084599
DOI/URL:

http://www.scopus.com/inward/record.url?eid=2-s2.0-3843051369&partnerID=40&md5=d63152e41ad48a832d5e4867447ac9f6
Notes: J. Biol. Chem. -- Cited By (since 1996):30 -- Export Date: 16 June 2014 -- CODEN: JBCHA -- Source: Scopus
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MSK Authors
  1. Jayakrishnan Nandakumar
    10 Nandakumar
  2. Stewart H Shuman
    546 Shuman
  3. Christopher D Lima
    103 Lima
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