Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains

RNA ligases participate in repair, splicing, and editing pathways that either reseal broken RNAs or alter their primary structure. Bacteriophage T4 RNA ligase (gp63) is the best-studied member of this class of enzymes, which includes yeast tRNA ligase and trypanosome RNA-editing ligases. Here, we identified another RNA ligase from the bacterial domain—a second RNA ligase (Rnl2) encoded by phage T4. Purified Rnl2 (gp24.1) catalyzes intramolecular and intermolecular RNA strand joining through ligase-adenylate and RNA-adenylate intermediates. Mutational analysis identifies amino acids required for the ligase-adenylation or phosphodiester synthesis steps of the ligation reaction. The catalytic residues of Rnl2 are located within nucleotidyl transferase motifs I, IV, and V that are conserved in DNA ligases and RNA capping enzymes. Rnl2 has scant amino acid similarity to T4 gp63. Rather, Rnl2 exemplifies a distinct ligase family, defined by variant motifs, that includes the trypanosome-editing ligases and a group of putative RNA ligases encoded by eukaryotic viruses (baculoviruses and an entomopoxvirus) and many species of archaea. These findings have implications for the evolution of covalent nucleotidyl transferases and virus-host dynamics based on RNA restriction and repair.

[1]  N. Cozzarelli,et al.  Bacteriophage T4 RNA ligase. Reaction intermediates and interaction of substrates. , 1977, The Journal of biological chemistry.

[2]  S. Shuman,et al.  Role of nucleotidyltransferase motifs I, III and IV in the catalysis of phosphodiester bond formation by Chlorella virus DNA ligase. , 2002, Nucleic acids research.

[3]  D. Teplow,et al.  Domain structure in yeast tRNA ligase. , 1990, Biochemistry.

[4]  S. Heaphy,et al.  Effect of single amino acid changes in the region of the adenylylation site of T4 RNA ligase. , 1987, Biochemistry.

[5]  H. Morris,et al.  Location of the adenylylation site in T4 RNA ligase. , 1985, European journal of biochemistry.

[6]  N. Cozzarelli,et al.  Bacteriophage T4 RNA ligase is gene 63 product, the protein that promotes tail fiber attachment to the baseplate. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[7]  B Sollner-Webb,et al.  Roles for ligases in the RNA editing complex of Trypanosoma brucei: band IV is needed for U‐deletion and RNA repair , 2001, The EMBO journal.

[8]  K. Kreuzer,et al.  Deletion of the essential gene 24 from the bacteriophage T4 genome. , 1993, Gene.

[9]  S. Shuman,et al.  Structure, mechanism, and evolution of the mRNA capping apparatus. , 2001, Progress in nucleic acid research and molecular biology.

[10]  G. Kaufmann,et al.  In vitro reconstitution of anticodon nuclease from components encoded by phage T4 and Escherichia coli CTr5X. , 1989, EMBO Journal.

[11]  K. Piller,et al.  The Two RNA Ligases of the Trypanosoma brucei RNA Editing Complex: Cloning the Essential Band IV Gene and Identifying the Band V Gene , 2001, Molecular and Cellular Biology.

[12]  P. Walter,et al.  Mechanism of non‐spliceosomal mRNA splicing in the unfolded protein response pathway , 1999, The EMBO journal.

[13]  S. Shuman,et al.  Vaccinia virus DNA ligase: specificity, fidelity, and inhibition. , 1995, Biochemistry.

[14]  Stewart Shuman,et al.  X-Ray Crystallography Reveals a Large Conformational Change during Guanyl Transfer by mRNA Capping Enzymes , 1997, Cell.

[15]  Z. Kelman,et al.  Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum. , 2000, Nucleic acids research.

[16]  R. Possee,et al.  The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. , 1994, Virology.

[17]  S. Shuman,et al.  RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases , 1995, Molecular microbiology.

[18]  D. Durantel,et al.  The pnk/pnl gene (ORF 86) of Autographa californica nucleopolyhedrovirus is a non-essential, immediate early gene. , 1998, The Journal of general virology.

[19]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[20]  S. Shuman,et al.  Crystal structure of eukaryotic DNA ligase-adenylate illuminates the mechanism of nick sensing and strand joining. , 2000, Molecular cell.

[21]  J. Hurwitz,et al.  Purification and properties of bacteriophage T4-induced RNA ligase. , 1972, Proceedings of the National Academy of Sciences of the United States of America.

[22]  C. Ho,et al.  Phylogeny of mRNA capping enzymes. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[23]  R. Levitz,et al.  Bacteriophage T4 anticodon nuclease, polynucleotide kinase and RNA ligase reprocess the host lysine tRNA. , 1987, The EMBO journal.

[24]  C. Ho,et al.  A yeast-like mRNA capping apparatus in Plasmodium falciparum , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[25]  S. Shuman,et al.  Chlorella virus DNA ligase: nick recognition and mutational analysis. , 1998, Nucleic acids research.

[26]  G. Kaufmann,et al.  Phage T4-coded Stp: double-edged effector of coupled DNA and tRNA-restriction systems. , 1995, Journal of molecular biology.

[27]  J. Hurwitz,et al.  Studies on ribonucleic acid ligase. Characterization of an adenosine triphosphate-inorganic pyrophosphate exchange reaction and demonstration of an enzyme-adenylate complex with T4 bacteriophage-induced enzyme. , 1974, The Journal of biological chemistry.

[28]  D. Wigley,et al.  Crystal Structure of an ATP-Dependent DNA Ligase from Bacteriophage T7 , 1996, Cell.

[29]  Michael T. McManus,et al.  Identification of candidate mitochondrial RNA editing ligases from Trypanosoma brucei. , 2001, RNA.

[30]  S. Shuman,et al.  Role of Nucleotidyl Transferase Motif V in Strand Joining byChlorella Virus DNA Ligase* , 2002, The Journal of Biological Chemistry.

[31]  R W Moyer,et al.  Complete genomic sequence of the Amsacta moorei entomopoxvirus: analysis and comparison with other poxviruses. , 2000, Virology.

[32]  S. Shuman,et al.  Covalent catalysis in nucleotidyl transfer reactions: essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Reza Salavati,et al.  An RNA Ligase Essential for RNA Editing and Survival of the Bloodstream Form of Trypanosoma brucei , 2001, Science.

[34]  S. Shuman,et al.  Mutational analysis of Chlorella virus DNA ligase: catalytic roles of domain I and motif VI. , 1998, Nucleic acids research.

[35]  H. Masaki,et al.  A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[36]  O. Uhlenbeck,et al.  2 T4 RNA Ligase , 1982 .