Characterization of a baculovirus enzyme with RNA ligase, polynucleotide 5'-kinase, and polynucleotide 3'-phosphatase activities.
The end-healing and end-sealing steps of the phage T4-induced RNA restriction-repair pathway are performed by two separate enzymes, a bifunctional polynucleotide 5'-kinase/3'-phosphatase and an ATP-dependent RNA ligase. Here we show that a single trifunctional baculovirus enzyme, RNA ligase 1 (Rnl1), catalyzes the identical set of RNA repair reactions. Three enzymatic activities of baculovirus Rnl1 are organized in a modular fashion within a 694-amino acid polypeptide consisting of an autonomous N-terminal RNA-specific ligase domain, Rnl1-(1-385), and a C-terminal kinase-phosphatase domain, Rnl1-(394-694). The ligase domain is itself composed of two functional units. The N-terminal module Rnl1-(1-270) contains essential nucleotidyltransferase motifs I, IV, and V and suffices for both enzyme adenylylation (step 1 of the ligation pathway) and phosphodiester bond formation at a preactivated RNA-adenylate end (step 3). The downstream module extending to residue 385 is required for ligation of a phosphorylated RNA substrate, suggesting that it is involved specifically in the second step of the end-joining pathway, the transfer of AMP from the ligase to the 5'-PO(4) end to form RNA-adenylate. The end-healing domain Rnl1-(394-694) consists of a proximal 5'-kinase module with an essential P-loop motif ((404)GSGKS(408)) and a distal 3'-phosphatase module with an essential acylphosphatase motif ((560)DLDGT(564)). Our findings have implications for the evolution of RNA repair systems and their potential roles in virus-host dynamics.
Martins
Alexandra
A
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
Stewart
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000201:enzymology*; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D009097:Multienzyme Complexes; D017856:Phosphotransferases (Phosphate Group Acceptor); D011117:Polyribonucleotide Nucleotidyltransferase / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D011130:RNA Ligase (ATP) / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D008670:Metals; D009097:Multienzyme Complexes; D014764:Viral Proteins; C081618:p35 protein, Nucleopolyhedrovirus; D012313:RNA; D000255:Adenosine Triphosphate; C022347:5'-phosphate-polyribonucleotide kinase; D017856:Phosphotransferases (Phosphate Group Acceptor); D011117:Polyribonucleotide Nucleotidyltransferase; D011130:RNA Ligase (ATP); C477285:bacteriophage T4 RNA ligase 1