title: Deinococcus radiodurans RNA ligase exemplifies a novel ligase clade with a distinctive N-terminal module that is important for 5'-PO4 nick sealing and ligase adenylylation but dispensable for phosphodiester formation at an adenylylated nick.
abstract: Deinococcus radiodurans RNA ligase (DraRnl) is a template-directed ligase that seals nicked duplexes in which the 3'-OH strand is RNA. DraRnl is a 342 amino acid polypeptide composed of a C-terminal adenylyltransferase domain fused to a distinctive 126 amino acid N-terminal module (a putative OB-fold). An alanine scan of the C domain identified 9 amino acids essential for nick ligation, which are located within nucleotidyltransferase motifs I, Ia, III, IIIa, IV and V. Seven mutants were dysfunctional by virtue of defects in ligase adenylylation: T163A, H167A, G168A, K186A, E230A, F281A and E305A. Four of these were also defective in phosphodiester formation at a preadenylylated nick: G168A, E230A, F281A and E305A. Two nick sealing-defective mutants were active in ligase adenylylation and sealing a preadenylylated nick, thereby implicating Ser185 and Lys326 in transfer of AMP from the enzyme to the nick 5'-PO(4). Whereas deletion of the N-terminal domain suppressed overall nick ligation and ligase adenylylation, it did not compromise sealing at a preadenylylated nick. Mutational analysis of 15 residues of the N domain identified Lys26, Gln31 and Arg79 as key constituents. Structure-activity relationships at the essential residues were determined via conservative substitutions. We propose that DraRnl typifies a new clade of polynucleotide ligases. DraRnl homologs are detected in several eukaryal proteomes.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Raymond
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000409:Alanine / Q000235:genetics; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000145:classification / Q000378:metabolism; D034301:Deinococcus / Q000201:enzymology*; D005057:Eukaryotic Cells / Q000201:enzymology; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D011130:RNA Ligase (ATP) / Q000737:chemistry* / Q000145:classification / Q000378:metabolism; D017385:Sequence Homology; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D012313:RNA; D011130:RNA Ligase (ATP); D000409:Alanine