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 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 Å 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 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 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.RNA repair | tRNA splicing | two-metal-ion catalysis R NA ligases join two RNA strands whose ends are produced by specific RNases in many biological processes during tRNA processing/splicing, antiphage, or unfolded protein response (1-3). Most widely studied are the 5′-Phosphate (5′-P) RNA ligases (4-6) that specifically catalyze the nucleophilic attack of a free 3′-hydroxyl on an activated 5′-P. However, these ligases cannot directly join two RNA strands ending with a 2′,3′-cyclic phosphate (RNA>p) and with a 5′-hydroxyl group. Before ligation, these strands require the action of a polynucleotide kinase (forming a 5′-P) and a phosphoesterase (generating a free 3′-OH) (6). The joining mechanism of 5′-P polynucleotide ligases involves a 3′-hydroxyl and an activated 5′-P end for internucleotide phosphodiester bond formation (7,8). The 5′-P activation enzymes include group I and II self-splicing introns, the spliceosomal apparatus, and DNA/RNA polymerases, as well as other nucleotidyl transferases (9-11). Similarly, the tRNA His guanylyltransferase ligates the 3′-OH of GTP to the 5′-terminus of tRNA through its adenylylation-activated 5′-P; this gives the appearance of reverse polarity of nucleotide addition relative to normal RNA polymerases (12).The 3′-Phosphate (3′-P) RNA ligase activities were identified three decades ago; they use a 3′-P as a donor for joining two RNAs without involvement of phosphorylation of the 5′-hydroxyl or dephosphorylation of the 2′,3′-cyclic phosphate (1...

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO 4 and 5′-OH ends

“…They support our initial hypothesis that RtcB binds its metal cofactor via "soft" enzymic ligands such as histidine nitrogens and a cysteine sulfur, such that "hard" metals like calcium and magnesium do not bind the active site, whereas soft metals such as zinc and cobalt do bind the active site (and out-compete manganese), but when so engaged are unable to sustain catalysis (1). This model is fortified by recent crystallographic evidence that RtcB binds two Mn 2+ ions in coordination complexes populated by soft ligands (20,21). A kinetic analysis of the isolated phosphodiester synthesis step catalyzed by the H337A mutant under conditions of enzyme excess is shown in Fig.…”

Characterization of the tRNA ligases of pathogenic fungi Aspergillus fumigatus and Coccidioides immitis

“…Fungal Trl1 enzymes are potential therapeutic targets because their domain structures and biochemical mechanisms are unique compared to the RtcB-type tRNA repair systems elaborated by metazoans, archaea, and many bacteria (Popow et al 2011;Tanaka and Shuman 2011;Tanaka et al 2011a;Englert et al 2012;Desai et al 2013;Maughan and Shuman 2015). RtcB is a GTP-dependent RNA ligase that splices 3 ′ -PO 4 and 5 ′ -OH ends via a novel chemical mechanism entailing the formation of covalent RtcB-(histidinyl)-GMP and RNA 3 ′ pp 5 ′G intermediates.…”

Structure of RNA 3′-phosphate cyclase bound to substrate RNA

“…The genes encoding RtcA and RtcB are localized into an operon in Escherichia coli and other bacterial taxa. The mechanism of nucleotidylation of histidine residues in RtcA and RtcB has been elucidated (Tanaka et al 2010;Chakravarty et al 2011;Englert et al 2012;Desai et al 2013); however, the location of the RNA binding sites and the mechanism of RNA 3 ′ -p nucleotidylation remain unknown for each enzyme. Moreover, how RtcA and RtcB avoid binding to the abundant RNA 3 ′ -OH termini in cellulo has remained an important unanswered question.…”

“…The RtcB active site is unique from that of RtcA with respect to its two metalbinding sites, which could help to coordinate and/or stabilize the 3 ′ -p and activated 3 ′ -p intermediate (Englert et al 2012;Desai et al 2013). RtcB has the difficult task of preventing intramolecular cyclization of the activated RNA intermediate (Chakravarty et al 2012), whereas RtcA has evolved to release this intermediate into solution (Filipowicz and Vicente 1990).…”