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.

Fig. 1.

RtcB is a GTP-dependent 3′-phosphate/5′-OH RNA ligase. RtcB splices a 3′-monophosphate RNA end to a 5′-OH end in a reaction that requires manganese and GTP. The dependence of ligation on GTP concentration is shown. Reaction mixtures containing 2 mM MnCl₂, 0.1 μM 20-mer RNA (depicted at bottom with the ³²P-label denoted by ●), 1 μM RtcB, and GTP as specified were incubated at 37 °C for 30 min. The labeled RNAs were resolved by PAGE. Intramolecular end-joining by RtcB converts the linear substrate to more rapidly migrating circular product.

Fig. 2.

Identification of a guanylylated RtcB tryptic peptide. (A) Top: Mass chromatogram of the mixture of tryptic peptides derived from RtcB after in vitro reaction with cold GTP. Bottom: Elution profile of a peptide with m/z of 732.92–732.94, consistent with a GMP adduct of the tryptic peptide ³²⁵GLGNEESFCSCSHGAGR³⁴¹ in which both cysteines were alkylated (denoted by ● over the C residues) and the net charge is +3. Middle: Elution profile of a peptide with m/z of 608.90–608.93, corresponding to the unguanylylated version of the same tryptic peptide, in which both cysteines were alkylated and the net charge is +3. (B) Mass spectrum of the guanylylated peptide M. The peaks corresponding to m/z values for the peptide designated M with net charges of +3 and +2 are indicated.

Fig. 3.

Identification of His337 as the site of guanylylation. (A) Depiction of the His337-guanylylated tryptic peptide. The sites of fragmentation of the guanylate moiety during tandem mass spectrometry are shown. Two unguanylylated peptide fragments, y11 and b13, detected in the MS/MS analysis are demarcated by brackets. (B) Nano-electrospray ionization MS/MS analysis of the guanylylated RtcB tryptic peptide. The unamplified peaks corresponding to the 17-aa peptides with +2 net charge after loss of GMP, guanosine (Guan), or guanine (Gua; alone or with water) are indicated. The 5× amplified region of the spectrum that includes GMP and peptide fragment y11²⁺ and the 20× amplified portion of the spectrum that includes peptide fragment b13 are demarcated by brackets. (C) Amplified spectra corresponding to peptide fragments y11²⁺ and b13.

Fig. 4.

His337 mutations abolish RtcB guanylylation and RNA ligation. (A) Aliquots (5 μg) of the wild-type and mutant RtcB preparations were analyzed by SDS/PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (kDa) of marker proteins are shown at left. (B) Label transfer from [α³²P]GTP to the wild-type and mutant RtcB proteins was analyzed by SDS/PAGE and autoradiography. (C) RNA ligase reaction mixtures containing 2 mM MnCl₂, 0.1 μM 20-mer RNA (depicted at bottom with the ³²P-labeled denoted by ●), 100 μM GTP, and 1 μM wild-type or mutant RtcB were incubated at 37 °C for 15 min. The labeled RNAs were analyzed by Urea-PAGE.

Fig. 5.

RtcB transfers GMP to a polynucleotide 3′-monophosphate end. (A) Reaction mixtures containing 2 mM MnCl₂, 10 μM [α³²P]GTP, 5 μM (50 pmol) RtcB, and 0, 1, or 5 pmol of an unlabeled 23-mer DNA with either a 3′-phosphate (3′-P) or a 3′-OH end were incubated for 30 min. The products were analyzed by Urea-PAGE and autoradiography. The 3′-P and 3′-OH DNAs are shown at bottom. (B) Kinetics of label transfer to the 3′-phosphate 23-mer DNA (5 pmol input DNA per aliquots analyzed). (C) The GMP-labeled 3′-phosphate 23-mer DNA was gel-purified and analyzed by PEI cellulose TLC, without further treatment, or after digestion with alkaline phosphatase (CIP), NPP, or P1, as indicated by +. The positions of cold GTP, GDP, and GMP markers are shown at right.

Fig. 6.

Polynucleotide-(3′)pp(5′)G is an intermediate in the ligation reaction. (A) A reaction mixture (90 μL) containing 2 mM MnCl₂, 100 μM GTP, 1 μM RtcB, and 0.1 μM _HORNA₁₉(dC)p (depicted with the 3′ deoxynucleoside shaded and the ³²P-label denoted by ●) was incubated at 37 °C. Aliquots (10 μL) containing 1 pmol of labeled _HORNA₁₉(dC)p substrate were withdrawn at the times specified and quenched immediately. The time 0 sample was withdrawn before adding the enzyme. Upper: Products were analyzed by urea-PAGE and visualized by autoradiography. The positions of the linear substrate and guanylylated intermediate are indicated on the left; the position of the ligated circle is indicated on the right. Lower: Kinetic profile of the reaction is plotted. A nonlinear regression curve fit of the data (in Prism) to a unidirectional two-step pathway is shown. (B) Kinetics of sealing of an all-RNA substrate (depicted with the ³²P-labeled denoted by ●) were assayed as described in A.