Kinetic and structural insights into the requirement of fungal tRNA ligase for a 2'-phosphate end

Abstract

Fungal RNA ligase (LIG) is an essential tRNA splicing enzyme that joins 3'-OH,2'-PO₄ and 5'-PO₄ RNA ends to form a 2'-PO₄,3'-5' phosphodiester splice junction. Sealing entails three divalent cation-dependent adenylate transfer steps. First, LIG reacts with ATP to form a covalent ligase-(lysyl-Nζ)-AMP intermediate and displace pyrophosphate. Second, LIG transfers AMP to the 5'-PO₄ RNA terminus to form an RNA-adenylate intermediate (A_5'pp_5'RNA). Third, LIG directs the attack of an RNA 3'-OH on AppRNA to form the splice junction and displace AMP. A defining feature of fungal LIG vis-à-vis canonical polynucleotide ligases is the requirement for a 2'-PO₄ to synthesize a 3'-5' phosphodiester bond. Fungal LIG consists of an N-terminal adenylyltransferase domain and a unique C-terminal domain. The C-domain of Chaetomium thermophilum LIG (CthLIG) engages a sulfate anion thought to be a mimetic of the terminal 2'-PO₄ Here, we interrogated the contributions of the C-domain and the conserved sulfate ligands (His227, Arg334, Arg337) to ligation of a pRNA_2'p substrate. We find that the C-domain is essential for end-joining but dispensable for ligase adenylylation. Mutations H227A, R334A, and R337A slowed the rate of step 2 RNA adenylation by 420-fold, 120-fold, and 60-fold, respectively, vis-à-vis wild-type CthLIG. An R334A-R337A double-mutation slowed step 2 by 580-fold. These results fortify the case for the strictly conserved His-Arg-Arg triad as the enforcer of the 2'-PO₄ end-specificity of fungal tRNA ligases and as a target for small molecule interdiction of fungal tRNA splicing.

Keywords: RNA end recognition; RNA repair; tRNA splicing.

FIGURE 1.

Domain structure and candidate 2′-PO₄ binding site of Chaetomium thermophilum LIG (CthLIG). (A) Illustration of the end-healing and end-sealing phases of the fungal tRNA splicing pathway. (B) Cartoon depiction of the domain organization of full-length 846-aa C. thermophilum Trl1 consisting of N-terminal ligase (LIG), central kinase (KIN), and C-terminal cyclic phosphodiesterase (CPD) domains. The N-terminal 407-aa segment comprising an autonomous LIG enzyme is the subject of the present study. (C) The tertiary structure of CthLIG is shown with the N-terminal adenylyltransferase module colored by secondary structure (magenta β strands and cyan α helices) and the unique C-terminal domain (aa 328-406) colored blue. ATP bound to the adenylyltransferase domain is depicted as a stick model. (D) View of the CthLIG active site depicting atomic contacts to ATP, two Mn²⁺ ions (green spheres), and metal-bound waters (red spheres). The image highlights a sulfate anion (stick model) bound to the C-domain, adjacent to the catalytic metal (Mn1), that is thought to mimic the 2′-PO₄ of the RNA substrate. Sulfate-binding residues His227, Arg334, and Arg337 were mutated to alanine in the present study.

FIGURE 2.

Structure-guided mutagenesis of CthLIG. (A) Aliquots (10 µg) of the purified full-length wild-type CthLIG, the indicated full-length alanine mutants, and the N-terminal adenylyltransferase domain (1–327) were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (kDa) of marker polypeptides are indicated on the left. (B) Ligation reaction mixtures (10 µL) containing 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 2 mM DTT, 10 mM MgCl₂, 100 µM ATP, 1 pmol (0.1 µM) ³²P-labeled 10-mer pRNA_2′p (shown at the top, with the ³²P-label indicated by filled circle [•]), and either no enzyme (lane –), or 1 pmol (0.1 µM) of the indicated CthLIG protein were incubated at 37°C for 30 min. The reactions were quenched with an equal volume of 95% formamide/50 mM EDTA, and the products were analyzed by electrophoresis (at 58 W constant power) through a 40 cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA. The radiolabeled RNAs were visualized by scanning the gel with a Typhoon FLA-7000 imaging device. The positions of the 5′-PO₄,2′-PO₄ RNA substrate (pRNAp), the 5-adenylylated intermediate (AppRNAp), and the 10-mer circle product of intramolecular ligation are indicated on the right. (C) Ligase adenylylation. Reaction mixtures (10 µL) containing 50 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 50 µM [α³²P]ATP, and 25 µM (250 pmol) of the indicated CthLIG protein were incubated at 37˚C for 5 min, then quenched with SDS, and analyzed by SDS-PAGE. A scan of the gel is shown.

FIGURE 3.

Analysis of the ligation reaction products. (A) Product analysis via treatment with CIP (calf intestine alkaline phosphatase). Ligation reactions containing 0.5 pmol 5′ ³²P-labeled pRNA_2′p and 10 pmol CthLIG (where indicated by +) were incubated at 37˚C for 30 min. The mixtures were then incubated for 10 min at 37˚C with 10 U CIP (purchased from NEB) where indicated by + above the lanes. The mixtures were quenched and then analyzed by urea-PAGE. (B) Product analysis via treatment with Tpt1 (RNA 2′-phosphotransferase). Ligation reactions containing 0.5 pmol 5′ ³²P-labeled pRNA_2′p and 10 pmol CthLIG (where indicated by +) were incubated at 37˚C for 30 min. The mixtures were then incubated for 30 min at 37˚C with 5 pmol Runella slithyformis Tpt1 (RsTpt1) and 1 mM NAD ⁺. The mixtures were quenched and then analyzed by urea-PAGE. The 5′ ³²P-phosphate is denoted by filled circle (•) in panels A and B. The ligated circular RNA with a 2′-OH splice junction formed after treatment with Tpt1 is indicated by the arrowhead at right in panels A and B. (C) Schematic of steps 2 and 3 of the ligation reaction and the species generated by treatment with CIP and Tpt1.

FIGURE 4.

ATP-independent ligation by CthLIG–AMP. (Top panel) Reaction mixtures (10 µL) containing 50 mM Tris-HCl, pH 8.0, 2 mM DTT, 5 mM MgCl₂, 1 pmol (0.1 µM) ³²P-labeled 10-mer pRNA_2’p, and CthLIG protein as specified were incubated at 37°C for 5 min and then quenched with formamide/EDTA. The mixtures were analyzed by urea-PAGE and the radiolabeled RNAs were visualized by scanning the gel. The positions of the pRNAp substrate, AppRNAp intermediate, and ligated circle product are indicated on the right. (Bottom panel) The extent of ligation is plotted as a function of input CthLIG. Each datum in the graph is the average of three independent titration experiments ± SEM. The error values are small, such that the error bars do not extent beyond the symbols.

FIGURE 5.

Kinetics of single-turnover ligation. (A) Individual replicate reaction mixtures (10 µL) containing 50 mM Tris-HCl, pH 8.0, 2 mM DTT, 5 mM MgCl₂, 0.1 µM 5′ ³²P-labeled 10-mer pRNAp substrate, and 2 µM wild-type CthLIG were incubated at 37˚C. The reactions were initiated by adding CthLIG to a prewarmed reaction mixture. The reactions were quenched after 5, 10, or 15 sec with an equal volume of 95% formamide/50 mM EDTA. The products were analyzed by urea-PAGE. The distribution of radiolabeled RNAs, either the individual AppRNAp and the ligated circle species (left panel) or the sum of AppRNAp and ligated circle (right panel) is plotted as a function of reaction time. Each datum in the graph is an average of five replicate reactions ± SEM. (B–E) Reaction mixtures (100 µL) containing 50 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 0.1 µM 5′ ³²P-labeled 10-mer pRNAp substrate, and 2 µM mutant CthLIG as specified were incubated at 37˚C. The reactions were initiated by adding mutant CthLIG to a prewarmed reaction mixture. Aliquots (10 µL) were withdrawn before adding enzyme (time 0) or at the times specified after enzyme addition and quenched immediately with an equal volume of formamide/EDTA. The products were analyzed by urea-PAGE. The distribution of radiolabeled RNAs is plotted as a function of reaction time. Each datum in the graphs is an average of three independent experiments ± SEM. The data in the graphs in the right column were fit by nonlinear regression to a one-phase association in Prism. The apparent step 2 (RNA adenylylation) rate constants are shown. The data in the graphs in the left column were fit by nonlinear regression to a unidirectional two-step kinetic mechanism. The Prism-calculated step 2 and step 3 (phosphodiester synthesis) rate constants are shown. Because there was no detectable AppRNAp formed during the H227A reaction, panel E includes only a one-phase association fit of the data in the graph in the right column.

FIGURE 6.

The His–Arg–Arg triad implicated in 2′-PO₄ binding is conserved in the tRNA ligases of fungal pathogens. The amino acid sequences in and flanking adenylyltransferase motifs III and V are aligned for the tRNA ligase enzymes of 18 human fungal pathogens designated as public health priorities by WHO. The defining glutamate of motif III and two lysines of motif V are shaded yellow. The histidine adjacent to motif III (corresponding to CthLIG His227) and the two arginine residues downstream from motif V (corresponding to CthLIG Arg334 and Arg337) are shaded cyan.