Dual Mechanisms whereby a Broken RNA End Assists the Catalysis of Its Repair by T4 RNA Ligase 2

It is concluded 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.

Abstract

T4 RNA ligase 2 (Rnl2) efficiently seals 3′-OH/5′-PO4RNA 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 ~102 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. T4 RNA ligase 2 (Rnl2) efficiently seals 3′-OH/5′-PO4RNA 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 ~102 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.