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J. Biol. Chem., Vol. 278, Issue 32, 29454-29462, August 8, 2003

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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^*

Li Kai Wang, C. Kiong Ho, Yi Pei and Stewart Shuman 

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021

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 Lys-99 N on the phosphorus of ATP. Mutation of Lys-99 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: Arg-54, Lys-75, Phe-77, Gly-102, Lys-119, Glu-227, Gly-228, Lys-240, and Lys-242. Seven of the essential residues are located within counterparts of conserved nucleotidyltransferase 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, Arg-54, Lys-75 and Phe-77, 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). Lys-75, Lys-240, and Lys-242 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 Arg-54 and Lys-119 abolished the overall RNA ligation reaction without affecting steps 1 and 3. Arg-54 and Lys-119 are thereby implicated as specific catalysts of the RNA adenylation reaction (step 2) of the ligation pathway.

Received for publication, April 24, 2003 , and in revised form, May 20, 2003.

^* This work was supported by National Institutes of Health Grant GM42498. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. E-mail: s-shuman{at}ski.mskcc.org.

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