Structure-guided mutational analysis of T4 RNA ligase 1.
T4 RNA ligase 1 (Rnl1) is a tRNA repair enzyme that circumvents an RNA-damaging host antiviral response. Whereas the three-step reaction scheme of Rnl1 is well established, the structural basis for catalysis has only recently been appreciated as mutational and crystallographic approaches have converged. Here we performed a structure-guided alanine scan of nine conserved residues, including side chains that either contact the ATP substrate via adenine (Leu179, Val230), the 2'-OH (Glu159), or the gamma phosphate (Tyr37) or coordinate divalent metal ions at the ATP alpha phosphate (Glu159, Tyr246) or beta phosphate (Asp272, Asp273). We thereby identified Glu159 and Tyr246 as essential for RNA sealing activity in vitro and for tRNA repair in vivo. Structure-activity relationships at Glu159 and Tyr246 were clarified by conservative substitutions. Eliminating the phosphate-binding Tyr37, and the magnesium-binding Asp272 and Asp273 side chains had little impact on sealing activity in vitro or in vivo, signifying that not all atomic interactions in the active site are critical for function. Analysis of mutational effects on individual steps of the ligation pathway underscored how different functional groups come into play during the ligase-adenylylation reaction versus the subsequent steps of RNA-adenylylation and phosphodiester formation. Moreover, the requirements for sealing exogenous preformed RNA-adenylate are more stringent than are those for sealing the RNA-adenylate intermediate formed in situ during ligation of a 5'-PO4 RNA.
Wang
Li Kai
LK
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Schwer
Beate
B
Shuman
Stewart
S
D000249:Adenosine Monophosphate / Q000737:chemistry; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D018698:Glutamic Acid / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011130:RNA Ligase (ATP) / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D014764:Viral Proteins; D018698:Glutamic Acid; D000249:Adenosine Monophosphate; D014443:Tyrosine; D011130:RNA Ligase (ATP); C477285:bacteriophage T4 RNA ligase 1; D000409:Alanine