RNA ligase RtcB splices 3′-phosphate and 5′-OH ends via covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates

Abstract

A cherished tenet of nucleic acid enzymology holds that synthesis of polynucleotide 3′-5′ phosphodiesters proceeds via the attack of a 3′-OH on a high-energy 5′ phosphoanhydride: either a nucleoside 5′-triphosphate in the case of RNA/DNA polymerases or an adenylylated intermediate A(5′)pp(5′)N-in the case of polynucleotide ligases. RtcB exemplifies a family of RNA ligases implicated in tRNA splicing and repair. Unlike classic ligases, RtcB seals broken RNAs with 3′-phosphate and 5′-OH ends. Here we show that RtcB executes a three-step ligation pathway entailing (i) reaction of His337 of the enzyme with GTP to form a covalent RtcB-(histidinyl-N)-GMP intermediate; (ii) transfer of guanylate to a polynucleotide 3′-phosphate to form a polynucleotide-(3′)pp(5′)G intermediate; and (iii) attack of a 5′-OH on the -N(3′)pp(5′)G end to form the splice junction. RtcB is structurally sui generis, and its chemical mechanism is unique. The wide distribution of RtcB proteins in bacteria, archaea, and metazoa raises the prospect of an alternative enzymology based on covalently activated 3′ ends. RNA or A(5′)pp(5′)DNA; and (iii) ligase catalyzes attack by a polynucleotide 3′-OH on A(5′)pp(5′)RNA/DNA to form a 3′-5′ phosphodiester bond and release AMP (1). The salient principle of classic ligase catalysis is that the high energy of the ATP phosphoanhydride bond is transferred via the enzyme to the polynucleotide 5′ end, thereby activating the 5′ end for phosphodiester synthesis, with AMP as a favorable leaving group. The shared chemical mechanism of classic RNA and DNA ligases is reflected in their conserved core tertiary structures and active sites (1).Classic ATP-dependent RNA ligases are encoded by diverse taxa in all three phylogenetic domains. In bacteria, fungi, and plants they function as components of multienzyme RNA repair pathways that heal and seal broken RNAs with 2′,3′ cyclic phosphate and 5′-OH ends (2-5). In the healing phase, the 2′,3′ cyclic phosphate end is hydrolyzed by a phosphoesterase enzyme to a 3′-OH, and the 5′-OH end is phosphorylated by a polynucleotide kinase enzyme to yield a 5′-monophosphate. The resulting 3′-OH and 5′-phosphate ends are then suitable for ATP-dependent sealing by RNA ligase. This "healing-and-sealing" pathway is responsible for tRNA splicing in fungi and plants (6, 7), for mRNA splicing in the fungal unfolded protein response (8), and for tRNA restriction repair during bacteriophage infection of Escherichia coli (9).An alternative "direct ligation" pathway for joining 2′,3′ cyclic phosphate and 5′-OH ends during mammalian tRNA splicing was discovered nearly 30 y ago (10-12), when it was shown that the 2′,3′ cyclic phosphate of the cleaved pre-tRNA is incorporated at the splice junction in the mature tRNA. (The junction phosphate in yeast tRNA splicing derives from the γ-phosphate of the NTP substrate for the 5′ kinase step.) Direct ligation languished until 2011, when three laboratories identified bacterial, archaeal, and mammalian RtcB proteins as RNA ligase enzymes capable of seali...

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

“…RtcB proteins were produced in E. coli and purified from soluble bacterial extracts as described (6). Protein concentrations were determined by the BioRad dye binding method with BSA as the standard.…”

“…Thus, the fate of pD12ppG reflects partitioning between forward phosphodiester synthesis and backward GMP transfer to RtcB, with the forward splicing step being favored under the conditions studied presently. The salient and instructive finding was that the RtcB H337A mutant, which is inert in joining 3′-PO 4 and 5′-OH ends (6), catalyzed quantitative splicing of the pD12ppG and HO DNA ends (Fig. 5A).…”

“…In light of the wide distribution of RtcB proteins in bacteria, archaea, and metazoa, we raised the prospect of an alternative enzymology based on covalently activated 3′-PO 4 ends (6).…”

“…RtcB executes a four-step pathway that requires GTP as an energy source and Mn 2+ as a cofactor (5)(6)(7). RtcB first reacts with GTP to form a covalent RtcB-(histidinyl 337 -N)-GMP intermediate.…”

Distinct Contributions of Enzymic Functional Groups to the 2′,3′-Cyclic Phosphodiesterase, 3′-Phosphate Guanylylation, and 3′-ppG/5′-OH Ligation Steps of the Escherichia coli RtcB Nucleic Acid Splicing Pathway

“…In principle, this issue can be addressed by identifying RtcB changes that cripple the endjoining pathway and then testing them for separations of function, i.e., whether such mutations selectively affect one or more of the chemical steps while sparing others. The utility of this approach was exemplified by an analysis of the H337A mutant of E. coli RtcB, which eliminates the site of covalent attachment of GMP to the enzyme, rendering the H337A mutant protein inert in RtcB guanylylation and, hence, formation of the polynucleotide-ppG intermediate (6). The instructive findings were that the H337A mutant was adept at joining DNAppG and HO DNA ends in a reaction that required Mn 2ϩ but not GTP (8), signifying that GTP and His337 play no essential role in the pathway downstream of the step of 3=-end activation.…”