Structure of 3′-PO4/5′-OH RNA ligase RtcB in complex with a 5′-OH oligonucleotide

RtcB enzymes comprise a widely distributed family of manganese- and GTP-dependent RNA repair enzymes that join 2',3'-cyclic phosphate ends to 5'-OH ends via RtcB-(histidinyl-N)-GMP, RNA 3'-phosphate, and RNA(3)'pp(5)'G intermediates. RtcB can ligate either 5'-OH RNA or 5'-OH DNA strands in vitro. The nucleic acid contacts of RtcB are uncharted. Here we report a 2.7 A crystal structure of Pyrococcus horikoshii RtcB in complex with a 6-mer 5'-OH DNA oligonucleotide (HO)A(1)pT(2)pG(3)pT(4)pC(5)pC(6), which reveals enzymic contacts of Asn202 to the terminal 5'-OH nucleophile; Arg238 to the A(1)pT(2) and T(2)pG(3) phosphates; Arg190 and Gln194 to the T(2)pG(3) phosphate; and an Arg190 p-cation interaction with the G(3) nucleobase. The structural insights affirm functional studies of E. coli RtcB that implicated the conserved counterpart of Arg238 in engagement of the 5'-OH strand for ligation. The essential active site Cys98 that coordinates two manganese ions is oxidized to cysteine sulfonic acid in our structure, raising the prospect that RtcB activity might be sensitive to modulation during oxidative stress.

Automated summary

RtcB enzymes are a family of manganese- and GTP-dependent RNA repair enzymes, capable of ligating both RNA and DNA strands. Structural analysis has revealed key nucleic acid contacts at the active site of RtcB, supporting functional studies on the enzyme.