A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation

Abstract

Archease is a 16-kDa protein that is conserved in all three domains of life. In diverse bacteria and archaea, the genes encoding Archease and the tRNA ligase RtcB are localized into an operon. Here we provide a rationale for this operon organization by showing that Archease and RtcB from Pyrococcus horikoshii function in tandem, with Archease altering the catalytic properties of the RNA ligase. RtcB catalyzes the GTP and Mn(II)-dependent joining of either 2',3'-cyclic phosphate or 3'-phosphate termini to 5'-hydroxyl termini. We find that catalytic concentrations of Archease are sufficient to activate RtcB, and that Archease accelerates both the RNA 3'-P guanylylation and ligation steps. In addition, we show that Archease can alter the NTP specificity of RtcB such that ATP, dGTP or ITP is used efficiently. Moreover, RtcB variants that have inactivating substitutions in the guanine-binding pocket can be rescued by the addition of Archease. We also present a 1.4 Å-resolution crystal structure of P. horikoshii Archease that reveals a metal-binding site consisting of conserved carboxylates located at the protein tip. Substitution of the Archease metal-binding residues drastically reduced Archease-dependent activation of RtcB. Thus, evolution has sought to co-express archease and rtcB by creating a tRNA splicing operon.

Figure 1.

The three nucleotidyl transfer steps of catalysis by RtcB, a putative tRNA splicing operon and the titration of Archease into RNA ligation reactions with RtcB. (A) The three nucleotidyl transfer steps of catalysis by RtcB are (1) RtcB guanylylation, (2) RNA 3′-P guanylylation and (3) phosphodiester bond formation. (B) The operon organization of rtcB and archease in diverse bacteria (Pelobacter propionicus, Synechococcus sp. JA-3-3Ab and Syntrophus aciditrophicus) and archaea (Halobacterium sp. NRC-1, Methanosaeta thermophila, P. horikoshii and Thermococcus kodakarensis). (C) RtcB-catalyzed RNA ligation reactions titrated with increasing concentrations of Archease, as specified. Reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl₂ (0.25 mM), GTP (0.10 mM), P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM). (RNA substrates are shown at top.) Reaction mixtures were incubated at 70°C for 30 min, and quenched with an equal volume of RNA gel-loading buffer. The reaction products were resolved by electrophoresis through an 18% w/v urea–polyacrylamide gel and visualized by fluorescence scanning of the FAM label. (D) Ligation product (nM) plotted versus Archease concentration (nM). Values are the mean ± SE for three separate experiments.

Figure 2.

Effect of Archease on the single-turnover rate of RNA ligation by RtcB. (A) RNA ligation reactions with RtcB alone or with the inclusion of 100 nM Archease. Reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl₂ (0.25 mM), GTP (0.10 mM), P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM). Reaction mixtures were incubated at 70°C, and aliquots were removed at the indicated times and quenched with an equal volume of RNA gel-loading buffer. (B) Plots of ligation product formation over time fitted to a single-exponential equation. Values are the mean ± SE for two separate experiments.

Figure 3.

Effects of Archease on the rate of RtcB-catalyzed guanylylation of RNA with a 2′-F/3′-P terminus. RtcB was pre-guanylylated by incubation with GTP and Mn(II), and the 3′ RNA fragment was not included to prevent ligation. (A) The guanylylation rate of a 2′-F/3′-P RNA terminus by RtcB alone or with the inclusion of 100 nM Archease. RNA guanylylation reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl₂ (0.25 mM), GTP (0.10 mM), P. horikoshii RtcB (5 μM) and RNA substrate (1.0 μM). (RNA substrate is shown at top.) Reaction mixtures were incubated at 70°C, and aliquots were removed at the indicated times and quenched with an equal volume of RNA gel-loading buffer. (B) Plots of RNA–ppG product formation over time fitted to a single-exponential equation. (C) Plots of RtcB-catalyzed RNA–ppG product formation over time in reactions that had the 2′-F/3′-P RNA substrate in excess (0.5 μM RtcB and 1.0 μM RNA substrate). Archease (100 nM) was added where indicated. Values in the plots are the mean ± SE for two separate experiments.

Figure 4.

RNA ligation reactions demonstrating the NTP cofactor specificity of RtcB and an active-site view of the RtcB–pG intermediate. (A) Reactions testing NTP cofactor specificity with RtcB alone or RtcB with 100 nM Archease. NTP cofactors were tested at 0.10 mM, and reaction mixtures were incubated at 70°C for 30 min. (B) Graph of the ligation product obtained for each NTP cofactor. Values are the mean ± SE for two separate experiments. (C) ATP-dependent RtcB-catalyzed RNA ligation reactions titrated with increasing concentrations of Archease, as specified. Reaction mixtures were incubated at 70°C for 20 min. Values are the mean ± SE for three separate experiments. (D) Crystal structure of the P. horikoshii RtcB–pG intermediate (PDB entry 4it0) illustrating the residues that contact the guanine nucleobase. (E–I) Michaelis–Menten plots of reaction rate versus NTP cofactor concentration for RtcB-catalyzed RNA ligation reactions under single-turnover conditions. Where indicated, Archease was included at a concentration of 100 nM for reactions with GTP, dGTP and ITP, while reactions with ATP included 800 nM Archease. Values are the mean ± SE for three separate experiments. (J) Single-turnover kinetics of ATP-dependent RNA ligation catalyzed by RtcB with the inclusion of Archease (800 nM). Values are the mean ± SE for two separate experiments. Ligation reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl₂ (0.25 mM), NTP as indicated, P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM).

Figure 5.

Crystal structure of P. horikoshii Archease. (A) A cartoon representation of the four Archease subunits per asymmetric unit with the Ca(II) ions represented as spheres. (B) The Ca(II) ion-binding site at the interface of subunits A and B. The Ca(II) ion-binding residues are depicted as sticks, and the Ca(II) ion and water molecules are shown as spheres. (C) Archease subunit A depicting the Ca(II) ion-binding site at the base of the N-terminal protrusion. A turn of 90° demonstrates the slenderness of the protein. (D) Electrostatic surface potential of Archease subunit A with blue and red indicating regions of positive and negative charge, respectively.

Figure 6.

Structure-guided mutagenesis of conserved Archease residues. (A, B) Archease variants with alanine substitutions were tested for their ability to activate RtcB in RNA ligation reactions with GTP (A) or ATP (B) as a cofactor. Reaction mixtures included 100 nM Archease where specified and were incubated at 70°C for 30 min. (C) Graph of the ligation product obtained for each Archease variant. Values are the mean ± SE for two separate experiments. Ligation reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl₂ (0.25 mM), GTP or ATP (0.10 mM), P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM).

Figure 7.

Effect of Archease on the activity of active-site variants of RtcB in RNA ligation assays with GTP or ATP as a cofactor. (A) Reactions with GTP (0.10 mM) as a cofactor. (B) Reactions with ATP (0.10 mM) as a cofactor. Reaction mixtures included 100 nM Archease where indicated, and were incubated at 70°C for 30 min. (C) Graph of the ligation product obtained for each RtcB variant. Values are the mean ± SE for two separate experiments. (D) ATP-dependent K480A RtcB-catalyzed RNA ligation reactions titrated with increasing concentrations of Archease, as specified. ATP was included at 0.10 mM, and reaction mixtures were incubated at 70°C for 15 min. Values are the mean ± SE for three separate experiments. (E) Michaelis–Menten plot of reaction rate versus ATP cofactor concentration for K480A RtcB-catalyzed RNA ligation reactions under single-turnover conditions. Values are the mean ± SE for three separate experiments. (F) Single-turnover kinetics of ATP-dependent RNA ligation catalyzed by K480A RtcB with the inclusion of Archease (800 nM). Values are the mean ± SE for two separate experiments. Ligation reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl₂ (0.25 mM), NTP as indicated, P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM).