Bacteriophage T4 RNA 2 (gp24.1) exemplifies a family of RNA found in all phylogenetic domains

C. Kiong Ho and Stewart Shuman*

Molecular Biology Program, The Sloan–Kettering Institute, New York, NY 10021

Edited by Nicholas R. Cozzarelli, University of California, Berkeley, CA, and approved July 5, 2002 (received for review March 28, 2002) RNA ligases participate in repair, splicing, and editing pathways that capping share a common tertiary structure composed of either reseal broken RNAs or alter their primary structure. Bacterio- five conserved motifs (I, III, IIIa, IV, and V) responsible for phage T4 RNA ligase (gp63) is the best-studied member of this class nucleotide binding and (21–23). It has been suggested that of enzymes, which includes yeast tRNA ligase and trypanosome DNA ligases and capping enzymes evolved from a common ances- RNA-editing ligases. Here, we identified another RNA ligase from the tral nucleotidyl , possibly from an ancient RNA strand- bacterial domain—a second RNA ligase (Rnl2) encoded by phage T4. joining . The structural basis for catalysis by RNA ligases is Purified Rnl2 (gp24.1) catalyzes intramolecular and intermolecular ill defined because few RNA ligase enzymes have been identified, RNA strand joining through ligase-adenylate and RNA-adenylate cloned, and studied. intermediates. Mutational analysis identifies amino acids required for Sequence-based searches for RNA ligases similar to T4 gp63 the ligase-adenylation or phosphodiester synthesis steps of the liga- identify yeast tRNA ligases (5) and a putative baculovirus ligase tion reaction. The catalytic residues of Rnl2 are located within nucle- (24) as the only credible homologs. Thus, gp63-like RNA ligases otidyl transferase motifs I, IV, and V that are conserved in DNA ligases have a narrow distribution in nature compared with the ubiquitous and RNA capping enzymes. Rnl2 has scant amino acid similarity to T4 DNA ligases. This situation may reflect the lack of selection gp63. Rather, Rnl2 exemplifies a distinct ligase family, defined by pressure to maintain the catalysis of RNA strand-transfer reactions variant motifs, that includes the trypanosome-editing ligases and a after the establishment of DNA genomes. Alternatively, RNA group of putative RNA ligases encoded by eukaryotic viruses (bacu- ligases with a gp63-like mechanism are widely distributed, but we loviruses and an entomopoxvirus) and many species of archaea. do not recognize them as such from sequence alone. For example, These findings have implications for the evolution of covalent nucle- the recently identified Trypanosoma brucei RNA-editing ligases otidyl and virus-host dynamics based on RNA restriction TbMP52 and TbMP48 (6–8) have little resemblance to the T4 gp63 and repair. RNA ligase, to the point that four iterations of a Psi-BLAST search with gp63 failed to identify the trypanosome RNA ligases and vice 4 RNA ligase (1) is the founding member of a family of RNA versa. Tend-joining enzymes involved in RNA repair, splicing, and Here, we identify and characterize another RNA-joining enzyme Ј Ј editing pathways (2–8). T4 RNA ligase joins 3 OH and 5 PO4 from the bacterial domain—a second and previously unrecognized RNA termini by means of three nucleotidyl transfer steps similar to RNA ligase encoded by phage T4. The additional T4 RNA ligase those of DNA ligases (9–11). Step 1 is the reaction of RNA ligase is the of gene 24.1 and will, henceforth, be referred to as with ATP to form a covalent ligase-(lysyl-N)–AMP intermediate. Rnl2 to distinguish it from the original ligase gp63, which we Ј In step 2, the AMP is transferred to a 5 PO4 RNA end to form an rename Rnl1. Rnl2 exemplifies a family of RNA ligases, defined by RNA-adenylate intermediate (AppRNA). In step 3, attack by an variant nucleotidyl transferase motifs, that includes proteins en- RNA 3Ј OH on RNA-adenylate seals the ends and releases AMP. coded by archaea, eukarya, and eukaryotic viruses. T4 RNA ligase can join two single-stranded RNA molecules without a complementary bridging polynucleotide. T4 RNA ligase Materials and Methods can also catalyze intramolecular ligation to form a covalently closed Recombinant T4 Rnl2. The gp24.1 ORF was amplified by PCR from RNA circle. RNA ligase has been a powerful tool in the synthesis T4 DNA (a gift of Ken Kreuzer, Duke University, Durham, NC) of RNAs of defined sequence, RNA 3Ј end modification, RNA 3Ј with primers designed to introduce an NdeI restriction site at the end-labeling, RNA sequencing, and structural analysis (11). start codon and a BamHI site 3Ј of the stop codon. The PCR During T4 infection in vivo, the RNA ligase, together with T4 product was digested with NdeI and BamHI and inserted into polynucleotide kinase (Pnk), performs an RNA repair function that pET16b (Novagen) to generate the plasmid pET-RNL2 encod- remodels and then seals broken tRNA ends. In Escherichia coli ing the T4 polypeptide fused to an N-terminal His-10 tag. Amino strains containing the prr locus, the host cell tRNALys is cleaved 5Ј acid substitution mutations were introduced into the ORF by to the wobble position by a T4-induced anticodon nuclease. If Pnk PCR with the two-stage overlap extension method (25). The and RNA ligase are not present, the synthesis of viral proteins is inserts of the WT and mutant pET-RNL2 plasmids were se- blocked by depletion of tRNALys, and the phage cannot replicate (2, quenced completely to exclude the acquisition of unwanted 12–14). This pathway represents an RNA-based restriction system changes during amplification and cloning. of host defense against a foreign invader. tRNA anticodon nuclease pET-RNL2 plasmids were transformed into E. coli BL21(DE3). ͞ systems are present in other pathogenic bacteria (14). The bacterial A 200-ml culture of E. coli BL21(DE3) pET-RNL2 was grown at BIOCHEMISTRY ͞ toxins colicins D and E5 are also anticodon nucleases that attack 37°C in LB medium containing 0.1 mg ml ampicillin until the A600 specific tRNAs (15). Thus, RNA repair enzymology has broad reached 0.4. The culture was adjusted to 0.4 mM isopropyl ␤-D- significance as a means to combat, or recover from, damage to thiogalactoside (IPTG), and incubation was continued at 37°C for essential RNA molecules. 3 h. Cells were harvested by centrifugation, and the pellet was stored T4 RNA ligase is a 374-aa polypeptide encoded by gene 63 (16). The site of covalent adenylation has been mapped to Lys-99 (17, 18), which is located within a conserved motif (KxDG) that defines This paper was submitted directly (Track II) to the PNAS office. a superfamily of covalent nucleotidyl transferases embracing DNA Abbreviation: NPV, nuclear polyhedrosis virus. ligases and mRNA capping enzymes (19, 20). DNA ligases and *To whom reprint requests should be addressed. E-mail: [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.192184699 PNAS ͉ October 1, 2002 ͉ vol. 99 ͉ no. 20 ͉ 12709–12714 Downloaded by guest on September 30, 2021 at Ϫ80°C. All subsequent procedures were performed at 4°C. Thawed bacteria were resuspended in 10 ml of buffer A [50 mM Tris⅐HCl, pH 7.5͞0.25 mM NaCl͞10% (wt͞vol) sucrose]. Lysozyme and Triton X-100 were added to final concentrations of 50 ␮g͞ml and a 0.1%, respectively. The lysate was sonicated to reduce viscosity and insoluble material was removed by centrifugation. The soluble extract was applied to a 1-ml column of Ni-NTA agarose (Qiagen, Chatsworth, CA) that had been equilibrated with buffer A containing 0.1% Triton X-100. The column was washed with 5 ml of the same buffer and then eluted stepwise with 2 ml of buffer B [50 mM Tris⅐HCl,pH8.0͞0.25 M NaCl͞10% (vol͞vol) glycerol] containing 0.05, 0.1, 0.2, and 0.5 M imidazole. The polypeptide compositions of the column fractions were monitored by SDS͞ PAGE. Rnl2 was recovered predominantly in the 0.1 M imidazole fraction, which contained 2–5 mg of protein. The WT and mutant Rnl2 preparations were stored at Ϫ80°C.

Adenylyltransferase Assay. Standard reaction mixtures (20 ␮l) con- taining 50 mM Tris acetate (pH 6.5), 5 mM DTT, 1 mM MgCl2,20 ␮M[␣-32P]ATP, and Rnl2 as specified were incubated for 5 min at 37°C. The reactions were quenched with SDS, and the products were analyzed by SDS͞PAGE. The ligase-[32P]AMP adduct was visualized by autoradiography of the dried gel and quantitated by scanning the gel with a PhosphorImager.

RNA Ligase Assay. An 18-mer oligoribonucleotide (5Ј-AUUC- CGAUAGUGACUACA) was 5Ј 32P-labeled by using T4 polynu- cleotide kinase and [␥-32P]ATP and then purified by electrophore- sis through a nondenaturing 20% polyacrylamide gel (26). RNA ligation reaction mixtures (10 ␮l) containing 50 mM Tris⅐acetate Ј 32 (pH 6.5), 5 mM DTT, 5 mM MgCl2, 1 pmol of 5 P-labeled 18-mer RNA, and ATP and Rnl2 as specified were incubated for 15 min at 22°C. The reactions were quenched by adding 5 ␮l of 90% form- amide͞20 mM EDTA. The samples were electrophoresed through an 18% polyacrylamide gel containing 7 M urea in 45 mM Tris⅐borate͞1 mM EDTA. The ligation products were visualized by autoradiography of the gel. Fig. 1. Rnl2-like family of RNA ligases. (A) The amino acid sequence of T4 Rnl2 from residues 1–227 is aligned to the sequences of the RNA-editing ligases Results TbMP52 and TbMP48 and putative ligases from the poxvirus AmEPV and bacu- Identification of a Second RNA Ligase Encoded by Bacteriophage T4. loviruses AcNPV and XcGV. (B) Alignment of Rnl2-like proteins from six species of Although T4 gp63 and the T. brucei RNA-editing ligases display archaea. Positions of side-chain identity͞similarity in all of the polypeptides little sequence similarity and, thereby, seem to comprise distinct included in the respective alignments are indicated by dots (Y). Nucleotidyl lineages, one can detect by visual inspection and manual alignment transferase motifs I, III, IIIa, IV, and V are highlighted in shaded boxes. the presence of nucleotidyl transferase motifs I, III, IIIa, IV, and V in both types of RNA ligases (6, 7). The nucleotidyl transferase ferase. The activities of Rnl2 were characterized in detail by using motifs of the T. brucei ligases are highlighted in Fig. 1. Motif I, which the 0.1 M imidazole eluate fraction. contains the active-site lysine, adheres to a consensus sequence KxHGxN in the trypanosome RNA ligases, which differs from the Adenylyltransferase Reaction. The adenylyltransferase activity dis- KxDGxR sequence characteristic of ATP-dependent DNA ligases played a bell-shaped pH profile with an optimum at pH 6.5 (Fig. and most mRNA capping enzymes (20, 27). A BLAST search with the 3A). Activity was virtually nil at pH Յ 5.0 or Ն 8.5. Rnl2 required T. brucei RNA ligases identified other proteins containing the a divalent cation to form the covalent adduct. MgCl2 and variant KxHGxN motif I sequence, including the putative RNA- MnCl2 supported optimal activity at 0.2–1 mM and 0.5–2mM editing ligase ortholog of Leishmania (7, 8) and a protein of concentrations, respectively (Fig. 3B). The yield of Rnl2-AMP unknown function (gp24.1) encoded by phage T4. The 334-aa T4 complex was proportional to ATP concentration from 0.2 to 5 ␮M gp24.1 polypeptide contains all five nucleotidyl transferase motifs in and reached saturation at 20 ␮M (Fig. 3C). Half-saturation was the usual order and with typical spacing between the motifs (Fig. achieved at 2 ␮M ATP. We calculated that Ϸ70% of the Rnl2 1A). Thus, we predicted that gp24.1 is a previously unappreciated protein was adenylated with 32P-AMP at saturating ATP. The second T4 RNA ligase, which we named Rnl2. remaining Ϸ30% of the Rnl2 preparation likely consists of pre- We expressed Rnl2 in E. coli as a His-10-tagged fusion and formed Rnl2-AMP intermediate (see below). purified the 42-kDa recombinant protein from a soluble bacterial The native size of Rnl2 was gauged by sedimentation through a extract by adsorption to Ni-agarose and step-elution with 50, 100, 15–30% glycerol gradient. Marker proteins catalase (248 kDa), and 200 mM imidazole (Fig. 2A). The adenylyltransferase activity BSA (66 kDa), and cytochrome c (13 kDa) were included as of recombinant Rnl2 was evinced by label transfer from [␣-32P]ATP internal standards. The adenylyltransferase activity sedimented as to the Rnl2 polypeptide to form a covalent enzyme-adenylate a single discrete peak between BSA and cytochrome c (Fig. 3D). adduct (Fig. 2B). Adenylyltransferase activity paralleled the elution The activity profile paralleled exactly the sedimentation profile of profile of the Rnl2 protein during Ni-agarose chromatography. We the Rnl2 polypeptide (not shown). We surmise that Rnl2 is a conclude that recombinant Rnl2 is a covalent nucleotidyl trans- monomer in solution.

12710 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.192184699 Ho and Shuman Downloaded by guest on September 30, 2021 Fig. 2. Purification and adenylyltransferase activity of T4 Rnl2. (A) Aliquots (10 ␮l) of the soluble lysate of isopropyl ␤-D-thiogalactoside-induced bacteria (L), the nickel-agarose flow-through (FT), and the indicated imidazole eluate fractions were analyzed by SDS͞PAGE. The gel was stained with Coomassie blue dye. The positions and sizes (kDa) of marker polypeptides are shown on the left. (B) Reaction mixtures (20 ␮l) containing 50 mM Tris⅐HCl (pH 8.0), 5 mM DTT, 5 mM 32 MgCl2, 0.17 ␮M[␣- P]ATP, and 1 ␮l of the indicated fractions were incubated for 5 min at 37°C. The reaction products were resolved by SDS͞PAGE. An autoradio- graph of the gel is shown.

Fig. 3. Characterization of the adenylyltransferase reaction. (A) pH depen- RNA Ligase Activity. Rnl2 was placed in reaction with a 5Ј 32P-labeled dence. Reaction mixtures (20 ␮l) containing 50 mM buffer (either Tris⅐acetate, pH 18-mer RNA oligonucleotide and magnesium in the presence or 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or Tris⅐HCl, pH 7.5, 8.0, 8.5, 9.0, 9.5), 5 mM DTT, 1 mM ␮ ␣ 32 absence of 1 mM ATP. In both cases, Rnl2 formed novel radio- MgCl2,20 M[ - P]ATP, and 200 ng of Rnl2 were incubated for 5 min at 37°C. labeled RNAs; however, the distribution of the products varied The extent of Rnl2-AMP formation is plotted as a function of pH. (B) Divalent cation dependence. Reaction mixtures (20 ␮l) containing 50 mM Tris⅐acetate (pH depending on whether ATP was present. When ATP was included, ␮ ␣ 32 Ϸ 6.5), 5 mM DTT, 20 M[ - P]ATP, 200 ng of Rnl2, and MgCl2 or MnCl2 as specified the predominant RNA product migrated 1 nt slower than the were incubated for 5 min at 37°C. Rnl2-AMP formation is plotted as a function of input 18-mer strand (Fig. 4A). This species, which was resistant to divalent cation concentration. (C) ATP dependence. Reaction mixtures (20 ␮l) alkaline phosphatase (not shown), corresponded to the RNA- containing 50 mM Tris⅐acetate (pH 6.5), 5 mM DTT, 1 mM MgCl2, 200 ng of Rnl2, adenylate (AppRNA) generated by AMP transfer from Rnl2-AMP and [␣-32P]ATP as specified were incubated for 5 min at 37°C. Rnl2-adenylate to the 5Ј end of the input 18-mer RNA. In the absence of ATP, the formation is plotted as a function of ATP concentration. (D) Glycerol gradient major product (denoted C1 in Fig. 4A) migrated Ϸ2 nt faster than sedimentation. An aliquot (50 ␮g) of Rnl2 was mixed with 50 ␮g each of catalase, BSA, and cytochrome c and the mixture was applied to a 4.8-ml 15–30% glycerol the input 18-mer strand. The C1 product, which was also resistant ⅐ to alkaline phosphatase, corresponds to a covalently closed 18-mer gradient containing 50 mM Tris HCl (pH 8.0), 2 mM DTT, 250 mM NaCl, 0.1% Triton X-100. The gradient was centrifuged at 50,000 rpm for 18 h at 4°Cina circle formed by intramolecular ligation. Ligation in the absence of Beckman SW50 rotor. Fractions were collected from the bottom of the tube. added ATP reflects the presence of preformed ligase-adenylate in Aliquots (1 ␮l) of the glycerol gradient fraction were assayed for adenylyltrans- the enzyme preparation. ferase activity. The activity profile is shown. The positions of the peak fractions of The Rnl2 reaction products also included higher molecular catalase, BSA, and cytochrome c are indicated by arrows. weight 32P-labeled species corresponding to a 36-mer linear dimer (L2), a 36-mer circular dimer (C2), and an adenylated 36-mer linear dimer (A-L2), respectively (Fig. 4A). As with the monomeric tion in the presence of ATP actually reflected Rnl2-catalyzed Ј Ј products, the formation of an adenylated linear dimer was favored joining of the 3 OH of ATP to the 5 PO4 of the 18-mer RNA to in the presence of ATP. At saturating levels of enzyme in reactions generate a slower-migrating product, pppApRNA. Two lines of containing ATP, 60% of the input was converted to experiments seem to exclude such a reaction. First, we found that RNA-adenylate, 10% to adenylated linear dimer, 10% to monomer other NTPs do not stimulate formation of the slower product at the circle, and 5% to dimer circle. In the absence of ATP, the circular expense of monomer circle, as was seen for ATP (not shown). If the Ј Ј dimer was the preferred product over the linear dimer (Fig. 4A). 3 OH of an NTP was being ligated to the 5 PO4, then Rnl2 would (Note that the linear RNA molecules migrated 1–2nt‘‘longer’’ than not be expected to have specificity for ATP as the 3Ј OH substrate. DNA strands of identical length and sequence. The circular RNAs In contrast, the model suggested above (whereby Rnl2 dissociates migrated aberrantly compared with linears.) after forming AppRNA and is trapped by covalent adenylylation) The inclusion of ATP promoted accumulation of AppRNAs and predicts that only ATP would stimulate formation of the slower suppressed formation of ligated circles. The likely explanation for species. Second, if the product was pppApRNA, then its electro- the ATP effect is that Rnl2 is prone to dissociate from the newly phoretic mobility should be altered by treatment with alkaline formed RNA-adenylate product of step 2, and that an immediate phosphatase (which would remove three 5Ј phosphates while 32

reaction with ATP to form ligase-adenylate precludes it from preserving the P in the RNA chain). We observed no shift in BIOCHEMISTRY rebinding to the RNA-adenylate for subsequent catalysis of strand mobility of the species we designated AppRNA after phosphatase joining. Similar ATP trapping effects leading to the accumulation treatment (not shown). of high levels of the adenylated nucleic acid intermediate have been The strand-joining reaction of T4 Rnl2 in the absence of ATP observed for DNA ligases when they react with a substrate con- required a divalent cation cofactor and was optimal at 0.5–5mM taining a 1-nt gap (26, 28). Sugino et al. (10) showed that catalysis MgCl2 (not shown). Although circles were the major product at of step 3 by T4 Rnl1 was inhibited by ATP and suggested that the each concentration tested (0.5, 1, 2, and 5 mM), the relative RNA intermediates dissociate from Rnl1 after RNA-adenylate is abundance of dimer circles was increased at 5 mM MgCl2. Similarly, formed. the formation of AppRNA in the presence of 1 mM ATP required It was conceivable that the change in the Rnl2 product distribu- a divalent cation and was optimal at 0.5–5 mM MgCl2; here also, the

Ho and Shuman PNAS ͉ October 1, 2002 ͉ vol. 99 ͉ no. 20 ͉ 12711 Downloaded by guest on September 30, 2021 Fig. 5. Mutational effects on adenylyltransferase activity. (A) Aliquots (4 ␮g) of the Ni-agarose preparations (0.1 M imidazole eluates) of wild-type Rnl2 and the indicated mutants were analyzed by SDS͞PAGE. The Coomassie blue- ␮ Fig. 4. RNA ligase activity. (A) Protein titration. Reaction mixtures (10 l) stained gel is shown. (B) Reaction mixtures contained 50 mM Tris⅐acetate (pH containing 50 mM Tris⅐acetate (pH 6.5), 5 mM DTT, 5 mM MgCl2, 1 pmol of 5Ј 32 6.5), 5 mM DTT, 1 mM MgCl2,20␮M[␣- P]ATP, and wild-type or mutant Rnl2, 32 ϩ P-labeled 18-mer RNA (pRNA), Rnl2 as specified, and either 1 mM ATP ( ATP) as specified. Ligase-adenylate formation is plotted as a function of input Rnl2 Ϫ or no ATP ( ATP) were incubated for 15 min at 22°C. (B) Kinetics. A reaction protein. mixture (100 ␮l) containing 50 mM Tris⅐acetate (pH 6.5), 5 mM DTT, 5 mM MgCl2, 10 pmol of pRNA substrate, and 800 ng of Rnl2 were incubated at 22°C. Aliquots ␮ (10 l) were withdrawn at the times indicated and quenched immediately with Reducing the pH to 5.0 or 4.5 completely suppressed the formation formamide-EDTA. (C) pH-dependence. Reaction mixtures (10 ␮l) containing 50 mM buffer (either Tris⅐acetate, pH 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0, or Tris⅐HCl, pH 7.0, of circles, but dramatically stimulated the formation of RNA- C 7.5, 8.0, 8.5, or 9.0), 5 mM DTT, 5 mM MgCl2, 1 pmol of pRNA substrate, and 80 ng adenylate (Fig. 4 ). This result implies that the step of phosphodi- of Rnl2 were incubated for 15 min at 22°C. Rnl2 was omitted from a control ester bond formation became rate-limiting at pH 4.5–5.0, such that reaction (–). The reaction products were analyzed by PAGE along with a mixture the normally evanescent RNA-adenylate intermediate accumu- of 5Ј 32P-labeled DNA oligonucleotide size markers (18-mer, 36-mer, and 60-mer; lated to high levels. Also, the transition from pH 5.5 to 5.0 resulted lane M in A). in the formation of adenylated linear dimers in lieu of nonactivated linear dimers (the Ϸ1-nt mobility difference between the A-L2 and L2 species is easily seen in Fig. 4C). Further reduction of the pH to adenylated linear dimer increased in abundance at 5 mM vs. 1 mM Յ4.0 abrogated all activity of Rnl2. Raising the pH to Ն7.5 MgCl2 (not shown). T4 Rnl2 failed to either ligate or adenylate a suppressed the intramolecular ligation reaction but did not diminish Ј 32 5 P-labeled 18-mer DNA oligonucleotide substrate under the the low amounts of linear dimers formed by intermolecular joining conditions that were permissive for RNA ligation and adenylation (Fig. 4C). (not shown). Analysis of the 32P-labeled reaction products after enzymatic digestion and polyethyleneimine-cellulose thin layer chromatogra- RNA-Adenylate Intermediate. A kinetic analysis of the strand-joining phy confirmed that 5Ј adenylated RNA was the major product reaction in the absence of ATP showed that RNA-adenylate was formed by Rnl2 in the presence of ATP under standard conditions the first product formed (Fig. 4B). RNA-adenylate persisted from (pH 6.5). All of the 32P-label in the substrate RNA (which migrated 32 0.5 to 2 min and then declined to undetectable levels by 5–10 min, as a smear near the chromatographic origin) was converted to Pi concomitant with decay of the 18-mer substrate and formation of by alkaline phosphatase, whereas most of the product generated by monomer and dimer circles (Fig. 4B). These results suggest that the Rnl2 with or without ATP resisted phosphatase digestion (Fig. 7, RNA-adenylate is a genuine intermediate along the Rnl2 ligation which is published as supporting information on the PNAS web site, reaction pathway. www.pnas.org). Treatment with nuclease P1 (which cleaves the 3Ј Additional evidence implicating RNA-adenylate as a reaction O–P bonds in the RNA) liberated 5Ј AMP as the predominant intermediate emerged from an analysis of pH effects on the labeled product from both the pRNA substrate and the ligation strand-joining reaction in the absence of ATP (Fig. 4C). The yields products formed in the absence of ATP (predominantly circles), but of the major circular products were optimal between pH 5.5 and 7.0. the P1 digestion product of the ligation products formed in the

12712 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.192184699 Ho and Shuman Downloaded by guest on September 30, 2021 The H37A change in motif I had no deleterious effect on formation of ligase-adenylate, whereas the introduction of Asp had a modest effect (2-fold decrement). The adenylation reaction was abrogated by the E204A change in motif IV and the K225A mutation in motif V. The K227A change in motif V reduced adenylyltransferase activity to 5% of the wild-type level (Fig. 5B). Mutational effects on RNA ligation in the presence and absence of ATP are shown in Fig. 6. Mutants K35A, E204A, and K225A failed to form either ligated RNAs or RNA-adenylate. This finding is in keeping with their inability to perform the initial ligase adenylation step in the reaction pathway upon which subsequent RNA transformations depend. K227A, which had feeble Rnl2 adenylation activity, formed only trace amounts of ligated RNA that required prolonged exposure of the gel to see (not shown). H37A, which was fully active in step 1 ligase adenylation, was Ј capable of transferring the adenylate to the 5 PO4 of RNA to form RNA-adenylate (in the presence or absence of ATP) but was selectively impaired at the step of phosphodiester bond formation (Fig. 6). The introduction of Asp at position 37 rectified the strand-joining defect of the H37A mutant. The spectrum of H37D reaction products resembled that of WT Rnl2 with regard to the predominance of the RNA-adenylate in the presence of ATP and of the circular monomer in the absence of ATP (Fig. 6). These findings show that functional groups within the nucleotidyl trans- ferase motifs of Rnl2 play essential roles at different steps of the RNA ligase reaction pathway. Discussion T4 Encodes a Second RNA Ligase. We have shown here that Rnl2, the product of T4 gene 24.1, is an RNA-specific polynucleotide Fig. 6. Mutational effects on RNA strand joining. Reaction mixtures contained ligase that catalyzes intramolecular and intermolecular RNA 50 mM Tris⅐acetate (pH 6.5), 5 mM DTT, 5 mM MgCl2, 1 pmol of pRNA substrate, strand joining through ligase-adenylate and RNA-adenylate 80 ng of WT or mutant Rnl2, and either 1 mM ATP (ϩ)ornoATP(Ϫ). The products intermediates. Cyclization of an 18-mer RNA was the favored were resolved by PAGE and visualized by autoradiography. 32P-labeled DNA outcome of the Rnl2 strand-joining reaction, rather than for- markers (18-mer, 36-mer, and 60-mer) were run in lane M. mation of linear multimers. This preference likely reflects proximity of the intramolecular 3Ј OH terminus to the . The same preference for cyclization is displayed by T4 Rnl1 with Ј presence of ATP was different and migrated slower that 5 AMP. substrates of similar size (10). RNA-adenylate was the predom- Combined digestion with alkaline phosphatase and nuclease P1 inant product formed by Rnl2 in the presence of 1 mM ATP, an 32 converted all of the label in the minus-ATP ligation products to Pi, effect likely caused by release of the enzyme from the step 2 yet the majority of the plus-ATP ligation product resisted this dual product and its immediate reaction with ATP to generate treatment and comigrated the material generated by nuclease P1 ligase-adenylate, as discussed above. Rnl2 also formed high alone (Fig. 7). This material corresponds to the phosphatase levels of the RNA-adenylate intermediate in the absence of ATP resistant 5Ј AppA dinucleotide liberated by digestion of AppRNAs when the reaction conditions were shifted to more acidic pH with nuclease P1. Quantitation of the chromatogram indicated that (4.5–5.0). Similar effects are observed for E. coli and T4 DNA 75% of the label in the plus-ATP product was converted to AppA, ligases, whereby the normally fleeting DNA-adenylate interme- a value that agrees with the 70% fraction of adenylated RNA diate can be trapped at acidic pH (29). Such findings suggest that products determined by PAGE (i.e., 60% adenylated monomer and the chemical mechanism of phosphodiester bond formation is 10% adenylated linear dimer). generally similar in RNA and DNA ligases, even though their substrate specificities (RNA vs. DNA, single-strand vs. double- Mutational Analysis of Rnl2 Identifies Residues Essential for Catalysis. strand) are different. The presence of the nucleotidyl transferase motifs in Rnl2 raised the To understand the structural requirements for RNA ligation, we question of whether and how they contribute to RNA ligase initiated an Ala-scanning mutational analysis of selected residues in function. To address this question, we introduced Ala substitutions the nucleotidyl transferase motifs of Rnl2. Our results implicate at Lys-35 and His-37 in motif I, Glu-204 in motif IV, and Lys-225 motif I residues Lys-35 and His-37 as essential catalysts of step 1 and Lys-227 in motif V. In addition, we replaced His-37 with Asp, (ligase adenylation) and step 3 (phosphodiester synthesis), respec- which is the side chain present at the equivalent position in motif tively. The requirement for Lys-35 of Rnl2 for overall ligation, and I of T4 Rnl1 and virtually all DNA ligases and RNA capping step 1 in particular, is consistent with mutational data for the

enzymes. equivalent lysine of T4 Rnl1 (18). Thus, we infer that Lys-35 is the BIOCHEMISTRY The K35A, H37A, H37D, E204A, K225A, and K227A mutants site of AMP attachment to Rnl2. The requirement for Rnl2 motif were expressed in bacteria and purified from soluble bacterial I residue His-37 is intriguing, given that most other polynucleotide lysates by Ni-agarose chromatography (Fig. 5A). The adenylyltrans- ligases have an Asp at this position. The H37A mutation caused a ferase activities were assayed by protein titration (Fig. 5B). The specific block to step 3 (without any apparent impact on step 1) that extent of ligase-adenylate formation by WT Rnl2 was proportional resulted in accumulation of RNA adenylate, from which we surmise to input protein up to 200 ng of protein. The K35A mutant was inert that His-37 is not essential for catalysis of step 2. Strand-joining over the same range. This result is consistent with the Rnl2 motif activity is restored by the H37D substitution, attesting to the I lysine being the site of covalent NMP attachment, as it is in other functional flexibility of this position of motif I in Rnl2. It is members of the polynucleotide ligase͞capping enzyme superfamily. noteworthy that whereas conservative mutations of the motif I

Ho and Shuman PNAS ͉ October 1, 2002 ͉ vol. 99 ͉ no. 20 ͉ 12713 Downloaded by guest on September 30, 2021 aspartate of T4 Rnl1 (to Asn, Ser, or Glu) also had no effect on step within the athropod vector may have played a role in the spread of 1, such changes abolished step 2 and step 3 of the ligation pathway the Rnl2-like ligases among these seemingly unrelated pathogens. (18). The motif I Asp is essential for step 2 catalysis by Chlorella Rnl2-like proteins are distributed widely among archaeal pro- virus DNA ligase and is also implicated in step 3 (30, 31). It would teomes, including those of Methanobacterium thermoautotrophi- seem that Rnl2 is less reliant on this motif I side chain for catalysis cum, Methanococcus jannaschii, Pyrococcus horikoshii, Pyrococcus of step 2 than is Rnl1 and DNA ligase. abyssi, Archaeoglobus fulgidus, and Aeropyrum pernix (Fig. 1B), as Here, we provide evidence that conserved residues in motifs IV well as Methanosarcina acetivorans, Methanosarcina mazei, and and V are essential for the activity of an RNA ligase. The corre- Methanopyrus kandleri (not shown). The archaeal Rnl2-like protein sponding side chains were shown to be essential for the activities of are of fairly uniform size (369–395 aa) and are significantly smaller DNA ligases (31, 32) and mRNA capping enzymes (33, 34). The than the archaeal ATP-dependent DNA ligases (37), which vary present results hint that the structural basis for nucleotidyl transfer from 556 to 619 aa in length. is at least partially conserved among RNA ligases, DNA ligases, and mRNA capping enzymes. Such conservation is consistent with the Possible Biological Roles of Rnl2 and Rnl2-Like Proteins. We can speculation that RNA-joining enzymes that evolved during a pri- deduce indirectly from the deletion analysis of gene 24 by Engman mordial RNA͞protein world are the ancestors of present-day DNA and Kreuzer (38) that the neighboring gp24.1 gene encoding Rnl2 ligases and mRNA capping enzymes (20). is nonessential for phage replication in a standard laboratory E. coli B strain. The RNA-joining activity of Rnl1 (gp63) is also not Rnl2 Exemplifies an RNA Ligase Family Found in All Phylogenetic essential for phage replication in E. coli B, but is required, together Domains. Although functional groups in motifs I, IV, and V are with Pnk, to reverse the tRNALys restriction defense mounted by essential for catalysis by Rnl2, there is little overall conservation of bacteria carrying the prr locus. Different bacterial strains in the wild primary structure between Rnl2 and the family of RNA ligases that may carry different tRNA restriction systems against which Rnl2 is includes T4 Rnl1 and fungal tRNA ligases. Instead, Rnl2 belongs to specifically suited to act. Alternatively, Rnl2 may participate in a distinct class of RNA ligases that includes the RNA editing ligases RNA-editing reactions akin to those performed by the Rnl2-like of kinetoplastid protozoa (Fig. 1A). A BLAST search for additional enzymes of trypanosomes. proteins related to T4 Rnl2 revealed a previously uncharacterized The coexistence of two RNA ligases in the same organism, with family of putative Rnl2-like ligases encoded by archaea and eu- complementary functions in related biological pathways, has clear karyotic DNA viruses (baculoviruses and a poxvirus; Fig. 1). The precedent in T. brucei, where the TbMP52 RNA ligase activity is additional members of the expanded Rnl2-like ligase family all specifically required for the editing pathway in which uridylates are contain nucleotidyl transferase motifs I, III, IIIa, IV, and V. The deleted from pre-mRNAs (39). The second ligase TbMP48 is distinctive and defining feature of this family is the strict conser- implicated in the pathway of uridylate insertion (39). The AcNPV vation of an asparagine located 5 amino acids downstream of the baculovirus is another case where two (putative) RNA ligases motif I lysine. Motif I in this family adheres to a consensus sequence inhabit the same biological niche. One of the AcNPV RNA ligases Kx[H,D,N]GxN. (the Orf86 product) resembles T4 Rnl1, whereas the second The eukaryotic viral branch of the Rnl2-like family is typified by baculovirus RNA ligase resembles T4 Rnl2. Given that the AcNPV the HE65 protein of the Autographa californica nuclear polyhedro- Orf86 protein seems to be a fusion of two enzymatic domains sis virus (NPV). AcNPV is the prototype of the baculoviruses, corresponding to T4 Pnk and T4 Rnl1, we speculate that the RNA which are nuclear DNA viruses that infect diverse arthropod hosts repair enzyme system of bacteriophage T4 is recapitulated in its (35). Other baculoviruses that encode an Rnl2-like protein include entirety in AcNPV. To extend the analogy further, we wonder Heliocoverpa armigera NPV, Bombyx mori NPV, and Xestia c- whether eukaryotic organisms can respond to virus infection by nigrum granulovirus (Fig. 1A). Little is known about the 553-aa triggering scission of essential RNAs, to which the virus must baculovirus HE65 protein, except that its gene is transcribed at early respond by catalyzing their repair. times during virus infection. A 524-aa Rnl2-like protein is also Archaea have a clear need for an RNA ligase to participate in the encoded by the Amsacta moorei entomopoxvirus (AmEPV), a removal of introns from their tRNAs. Archaea do not encode a cytoplasmic poxvirus that infects caterpillars (36). It is notable that recognizable homolog of yeast tRNA ligase, even though they do Rnl2-like putative ligases in the eukaryotic domain are apparently possess a tRNA splicing endonuclease that resembles the fungal restricted to viruses (baculo and entomopox) and protozoa endonuclease (3). The Rnl2-like proteins of archaea are plausible (Trypanosoma and Leishmania) that spend all or part of their life candidates for a ligase component of the archaeal tRNA splicing cycles in an arthropod host. We speculate that lateral gene transfer machinery.

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