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 Journal Article
| Authors: | Wang, L. K.; Ho, C. K.; Pei, Y.; Shuman, S. |
| Article Title: | 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 |
| Abstract: | 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 Nζ on the α 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. |
| Keywords: | unclassified drug; mutation; nonhuman; enzyme activity; structure activity relation; structure-activity relationship; mutational analysis; rna; dna; amino acid sequence; molecular sequence data; sequence homology, amino acid; saccharomyces cerevisiae; protein structure, tertiary; binding sites; dna mutational analysis; alanine; adenosine triphosphate; transfer rna; rna, transfer; biochemistry; lysine; rna ligase (atp); arginine; phenylalanine; amino acid motifs; catalyst activity; viral proteins; adenylation; virus mutation; bacteria; chemical bonds; rna ligase; saccharomyces; bacteriophage t4; substitution reactions; adenosinetriphosphate; unidentified baculovirus; priority journal; article; rna ligase 1; electrophoresis, agar gel |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 278 |
| Issue: | 32 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2003-08-08 |
| Start Page: | 29454 |
| End Page: | 29462 |
| Language: | English |
| DOI: | 10.1074/jbc.M304320200 |
| PUBMED: | 12766156 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 12 September 2014 -- Source: Scopus |
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