Structural and mechanistic insights into guanylylation of RNA-splicing ligase RtcB joining RNA between 3′-terminal phosphate and 5′-OH

Abstract

The RtcB protein has recently been identified as a 3′-phosphate RNA ligase that directly joins an RNA strand ending with a 2′,3′-cyclic phosphate to the 5′-hydroxyl group of another RNA strand in a GTP/Mn 2+-dependent reaction. Here, we report two crystal structures of Pyrococcus horikoshii RNA-splicing ligase RtcB in complex with Mn2+ alone (RtcB/ Mn2+) and together with a covalently bound GMP (RtcB-GMP/Mn2+). The RtcB/ Mn2+ structure (at 1.6 Åresolution) shows two Mn2+ ions at the active site, and an array of sulfate ions nearby that indicate the binding sites of the RNA phosphate backbone. The structure of the RtcB-GMP/Mn2+ complex (at 2.3 Å resolution) reveals the detailed geometry of guanylylation of histidine 404. The critical roles of the key residues involved in the binding of the two Mn2+ ions, the four sulfates, and GMP are validated in extensive mutagenesis and biochemical experiments, which also provide a thorough characterization for the three steps of the RtcB ligation pathway: (i ) guanylylation of the enzyme, (ii ) guanylyl-transfer to the RNA substrate, and (iii) overall ligation. These results demonstrate that the enzyme's sub-strate-induced GTP binding site and the putative reactive RNA ends are in the vicinity of the binuclear Mn2+ active center, which provides detailed insight into how the enzyme-bound GMP is tansferred to the 3′-phosphate of the RNA substrate for activation and subsequent nucleophilic attack by the 5′-hydroxyl of the second RNA substrate, resulting in the ligated product and release of GMP.