4dwq
RNA ligase RtcB-GMP/Mn(2+) complex
Structural highlights
FunctionRTCB_PYRHO tRNA-splicing ligase that acts by directly joining spliced tRNA halves to mature-sized tRNAs by incorporating the precursor-derived splice junction phosphate into the mature tRNA as a canonical 3',5'-phosphodiester. May act as a RNA ligase with broad substrate specificity, and may function toward other RNAs (By similarity). Publication Abstract from PubMedThe 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 Mn(2+) alone (RtcB/ Mn(2+)) and together with a covalently bound GMP (RtcB-GMP/Mn(2+)). The RtcB/ Mn(2+) structure (at 1.6 A resolution) shows two Mn(2+) 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/Mn(2+) complex (at 2.3 A resolution) reveals the detailed geometry of guanylylation of histidine 404. The critical roles of the key residues involved in the binding of the two Mn(2+) 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 substrate-induced GTP binding site and the putative reactive RNA ends are in the vicinity of the binuclear Mn(2+) 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. Structural and mechanistic insights into guanylylation of RNA-splicing ligase RtcB joining RNA between 3'-terminal phosphate and 5'-OH.,Englert M, Xia S, Okada C, Nakamura A, Tanavde V, Yao M, Eom SH, Konigsberg WH, Soll D, Wang J Proc Natl Acad Sci U S A. 2012 Sep 18;109(38):15235-40. Epub 2012 Sep 4. PMID:22949672[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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