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. 2019 Dec 2;47(21):11430-11440.
doi: 10.1093/nar/gkz965.

Branch site bulge conformations in domain 6 determine functional sugar puckers in group II intron splicing

Raphael Plangger  1 , Michael Andreas Juen  1 , Thomas Philipp Hoernes  2 , Felix Nußbaumer  1 , Johannes Kremser  1 , Elisabeth Strebitzer  1 , David Klingler  1 , Kevin Erharter  1 , Martin Tollinger  1 , Matthias David Erlacher  2 , Christoph Kreutz  1
Affiliations

Branch site bulge conformations in domain 6 determine functional sugar puckers in group II intron splicing

Raphael Plangger et al. Nucleic Acids Res. .

Abstract

Although group II intron ribozymes are intensively studied the question how structural dynamics affects splicing catalysis has remained elusive. We report for the first time that the group II intron domain 6 exists in a secondary structure equilibrium between a single- and a two-nucleotide bulge conformation, which is directly linked to a switch between sugar puckers of the branch site adenosine. Our study determined a functional sugar pucker equilibrium between the transesterification active C2'-endo conformation of the branch site adenosine in the 1nt bulge and an inactive C3'-endo state in the 2nt bulge fold, allowing the group II intron to switch its activity from the branching to the exon ligation step. Our detailed NMR spectroscopic investigation identified magnesium (II) ions and the branching reaction as regulators of the equilibrium populations. The tuneable secondary structure/sugar pucker equilibrium supports a conformational selection mechanism to up- and downregulate catalytically active and inactive states of the branch site adenosine to orchestrate the multi-step splicing process. The conformational dynamics of group II intron domain 6 is also proposed to be a key aspect for the directionality selection in reversible splicing.

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Figures

Figure 1.
Figure 1.
Group II intron splicing pathways, conformational rearrangement between step 1 and 2, D56 construct used in this study. (A) Branching pathway: Between step 1 and 2 a secondary structure switch is postulated by Costa and co-workers (16). (B) Hydrolysis pathway: Instead of the branch site adenosine a water molecule acts as the nucleophile in the first step of splicing. (C) Schematic secondary structure representation of domain 6 of the chimeric Oc19 construct, in which the branching pathway is activated. A rearrangement from a single-bulge (1B) to a two-bulge (2B) between the branching step and the exon ligation step was postulated by Costa et al. (16). Branch-site adenosine is highlighted in red and blue. (D) Trans-active D56 RNA in the 1B and 2B fold state with numbering scheme. Branch-site adenosine is highlighted in red and blue.
Figure 2.
Figure 2.
Secondary structure equilibrium in D56. (A) The 61nt D56 RNA with 13C1′-labels highlighted in red (1nt bulge fold - 1B) and blue (2nt bulge fold - 2B). Equilibrium populations of 1B and 2B are given. (B) Imino proton spectrum of the 61nt D56 RNA. The two NH1 resonances for G16 were assigned by site-specific 15N1-G labeling and confirm the slow exchange process in D6. (C) 13C longitudinal exchange spectrum with 13C1′-labelled C18, C19, U22, A23 and U24 at 25°C and 50 ms mixing time. Assignments are given. (D) Exchange rate constants in s−1 determined from 13C longitudinal exchange NMR experiments. Red forward rate constant kf, blue backward rate constant kB and grey exchange rate kex = kf + kb.
Figure 3.
Figure 3.
Regulators of the 1B–2B fold equilibrium populations. (A) Schematic summary of factors tuning the equilibrium populations. The addition of MgCl2 and branched RNA formation lead to a preference of 2B, whereas the addition of ethylenediaminetetraacetic acid (EDTA), a chelating agent for magnesium (II) ions, favours fold 1B. (B) Schematic structure of the 2nt bulge in 2B. The 2nt bulge allows a higher degree of conformational flexibility in the branched state. (C) G16 N1H imino resonance a function of MgCl2 and EDTA additions. (D) HMQC spectrum of the A4 1H8–13C8-correlation as a function of MgCl2 additions. (E) G16 N1H imino resonance in branched and unbranched state. (F) HMQC spectrum of the A4 1H8–13C8-correlation in the branched state with 25 eq. MgCl2 added. (G) Bar plots of equilibrium populations at various MgCl2 and EDTA concentrations. (H) Bar plots of equilibrium populations in branched state with and without magnesium (II) ions.
Figure 4.
Figure 4.
Sugar puckers in 1B and 2B. (A) Secondary structure representation of the G6C/C21G mutant populating only state 1B. Mutations are highlighted in red. U12 and A23 also highlighted in red carry a 13C1′ label. (B) H1′(C1′)H2′ experiment for the determination of the sugar pucker. For both residues a cross peak was observed. (C) Schematic representation of sugar pucker equilibrium of A23 in state 1B. A 3JH1′H2′ = 4.6 Hz scalar coupling constant was determined for A23 translating in a 54/46 C3′/C2′-endo sugar pucker equilibrium. (D) Secondary structure representation of the G6C/C20G mutant populating only state 2B. Mutations are highlighted in blue. U12 and A23 also highlighted in blue carry a 13C1′ label. (E) H1′(C1′)H2′ experiment for the determination of sugar pucker. Only for U12 a cross peak was observed. (F) Schematic representation of sugar pucker equilibrium of A23 in state 2B. The 3JH1′H2′ Hz scalar coupling constant could not be determined for A23 translating in a purely C3′-endo sugar pucker.
Figure 5.
Figure 5.
A linked secondary structure and sugar pucker equilibrium for the selection of catalytically relevant states. The sugar pucker equilibrium of the branch site adenosine and the secondary structure distributions during the two steps of group II intron catalysis are given. Inactive folds for the forward self-splicing pathway are shown transparent. These states are the active states in the reverse splicing pathway. Our data strongly supports a conformational selection mechanism in group II intron catalysis to guarantee the availability of reaction competent states for either the forward or backward reaction pathway.
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