Structure-function analysis of T4 RNA ligase 2.
Bacteriophage T4 RNA ligase 2 (Rnl2) exemplifies a polynucleotide ligase family that includes the trypanosome RNA-editing ligases and putative RNA ligases encoded by eukaryotic viruses and archaea. Here we analyzed 12 individual amino acids of Rnl2 that were identified by alanine scanning as essential for strand joining. We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of ligase adenylylation and phosphodiester bond formation. The essential residues of Rnl2 are located within conserved motifs that define a superfamily of nucleotidyl transferases that act via enzyme-(lysyl-N)-NMP intermediates. Our mutagenesis results underscore a shared active site architecture in Rnl2-like ligases, DNA ligases, and mRNA capping enzymes. They also highlight two essential signature residues, Glu(34) and Asn(40), that flank the active site lysine nucleophile (Lys(35)) and are unique to the Rnl2-like ligase family.
Yin
Shenmin
S
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Ho
C Kiong
CK
Shuman
Stewart
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D011118:Polynucleotide Ligases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D012313:RNA / Q000378:metabolism; D011130:RNA Ligase (ATP); D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*
D011994:Recombinant Proteins; D014764:Viral Proteins; D012313:RNA; D000255:Adenosine Triphosphate; D011118:Polynucleotide Ligases; D011130:RNA Ligase (ATP); C467724:bacteriophage T4 RNA ligase 2; D008239:Lysine; D000409:Alanine