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Papers In Press, published online ahead of print May 24, 2003
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021
Corresponding Author: s-shuman{at}ski.mskcc.org
T4 RNA ligase 1 (Rnl1) exemplifies an ATP-dependent RNA ligase family that includes fungal tRNA ligase (Trl1) and a putative baculovirus RNA ligase. Rnl1 acts via a covalent enzyme–AMP intermediate generated by attack of Lys99 Nz on the a phosphorus of ATP. Mutation of Lys99 abolishes ligase activity. Here we tested the effects of alanine mutations at 19 conserved positions in Rnl1 and thereby identified 9 new residues essential for ligase activity: Arg54, Lys75, Phe77, Gly102, Lys119, Glu227, Gly228, Lys240, and Lys242. Seven of the essential residues are located within counterparts of conserved nucleotidyl transferase motifs I (99KEDG102), Ia (118SK119), IV (227EGYVA231), and V (238HFKIK242) that comprise the active sites of DNA ligases, RNA capping enzymes and T4 RNA ligase 2. Three other essential residues, Arg54, Lys75 and Phe77, are located upstream of the AMP attachment site within a conserved domain unique to the Rnl1-like ligase family. We infer a shared evolutionary history and active site architecture in Rnl1 (a tRNA repair enzyme) and Trl1 (a tRNA splicing enzyme). We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of Rnl1 adenylylation (step 1) and phosphodiester bond formation (step 3). Lys75, Lys240 and Lys242 were found to be essential for step 1 and overall ligation of 5’-phosphorylated RNA, but not for phosphodiester bond formation. These results suggest that the composition of the Rnl1 active site is different during steps 1 and 3. Mutations at Arg54 and Lys119 abolished the overall RNA ligation reaction without affecting steps 1 and 3. Arg54 and Lys119 are thereby implicated as specific catalysts of the RNA adenylation reaction (step 2) of the ligation pathway.
J. Biol. Chem, 10.1074/jbc.M304320200
Submitted on April 24, 2003
Revised on May 20, 2003
Accepted on May 24, 2003
Mutational analysis of bacteriophage T4 RNA ligase 1: Different functional groups are required for the nucleotidyl transfer and phosphodiester bond formation steps of the ligation reaction
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