Dual mechanisms whereby a broken RNA end assists the catalysis of its repair by T4 RNA ligase 2.
T4 RNA ligase 2 (Rnl2) efficiently seals 3'-OH/5'-PO4 RNA nicks via three nucleotidyl transfer steps. Here we show that the terminal 3'-OH at the nick accelerates the second step of the ligase pathway (adenylylation of the 5'-PO4 strand) by a factor of 1000, even though the 3'-OH is not chemically transformed during the reaction. Also, the terminal 2'-OH at the nick accelerates the third step (attack of the 3'-OH on the 5'-adenylated strand to form a phosphodiester) by a factor of 25-35, even though the 2'-OH is not chemically reactive. His-37 of Rnl2 is uniquely required for step 3, providing a approximately 10(2) rate acceleration. Biochemical epistasis experiments show that His-37 and the RNA 2'-OH act independently. We conclude that the broken RNA end promotes catalysis of its own repair by Rnl2 via two mechanisms, one of which (enhancement of step 3 by the 2'-OH) is specific to RNA ligation. Substrate-assisted catalysis provides a potential biochemical checkpoint during nucleic acid repair.
Nandakumar
Jayakrishnan
J
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
Stewart
S
D002384:Catalysis; D004843:Epistasis, Genetic; D004952:Esters; D009154:Mutation; D011130:RNA Ligase (ATP) / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004952:Esters; D012367:RNA, Viral; D014764:Viral Proteins; D011130:RNA Ligase (ATP); C467724:bacteriophage T4 RNA ligase 2