Visualizing group II intron dynamics between the first and second steps of splicing

Figures

• 

  Fig. 1. Kinetics of intron mutants. a Schematics of the splicing reaction and sketch of the chemical mechanism of catalysis by group II introns. k1 is the rate constant of the first and k2 of the second step of splicing. Kinetic rate constants of all constructs are reported in Supplementary Table 2. Black arrows indicate nucleophilic attacks; gray dotted lines indicate interactions between oxygen atoms of the scissile phosphate groups and catalytic metal ions; Nuc indicates the reaction nucleophile. b Representative splicing kinetics of wild-type intron and A, G, and U mutants. Precursors are indicated as 5e-I-3e (nt length in parenthesis). Intermediate (I-3e) and linear intron (I) migrate as double or triple bands because of cryptic cleavage sites, as explained previously16. c Evolution of the populations of precursor (5e-I-3e, left panel), intermediate (I-3e, middle panel), and linear intron (I, right panel) over time. Error bars represent standard errors of the mean (s.e.m.) calculated from n = 3 independent experiments. Source data are provided as a Source Data file.

• 

  Fig. 2. Crystal structures of the intron in potassium and magnesium. a From left to right: Active site of wild type (WT; PDB id: 4FAR), G-mutant, and U-mutant in the state following 5′-exon hydrolysis. The intron is depicted as a cartoon representation in light green, the 5′-exon is in gray, and the mutated triplex is in blue (black dotted lines indicate H-bonds forming the triple helix). b Fo − Fc simulated-annealing electron density omit-maps calculated by omitting J2/3 residues (nt 287–289) and the M1-M2-K1-K2 metal cluster from each of the structures depicted in (a). Positive electron density peaks within 3 Å from the omitted atoms are depicted as blue mesh at a contour level of 3 σ. The toggled conformation of G288 (from PDB id: 4FAX) is depicted as a semi-transparent light blue cartoon representation for reference. c Fo − Fc simulated-annealing electron density omit-maps calculated by omitting G1 from each of the structures depicted in (a). Positive electron density peaks within 3 Å from the omitted atoms are depicted as blue mesh at a contour level of 3 σ. G1 is unresolved in the structure of the U-mutant. d Superposition of the active sites of the structures depicted in (a). e…

• 

  Fig. 3. Crystal structures of the intron in sodium and magnesium. a From left to right: Active site of wild type (WT; PDB id: 4FAX), G-mutant, and U-mutant. Active site elements are depicted as in Fig. 2a. b Fo − Fc simulated-annealing electron density omit-maps calculated by omitting J2/3 residues (nt 287–289) and the M1–M2–K1–K2 metal cluster from each of the structures depicted in (a). Positive electron density peaks within 3 Å from the omitted atoms are depicted as blue mesh at a contour level of 3 σ. For the wild-type structure (left), the triple helix conformation of G288 is depicted as semi-transparent light blue cartoon representation, the M1–M2 metals as semi-transparent yellow spheres, and the K1–K2 metals as semi-transparent violet spheres (all using coordinates from PDB id: 4FAR). Analogously, the toggled conformation of G288 and unresolved metals of the cluster are depicted as semi-transparent representations for the G- and U-mutant (middle and right panels, using coordinates from PDB id: 4FAX for G288 and 4FAR for unresolved metals). c Superposition of the active sites of the structures depicted in (a).

• 

  Fig. 4. Importance of the K1 interaction with N7G288. a Changes during MD simulations of the pre-hydrolytic state for key structural descriptors dK1-N7G288 (green trace) and dM1-M2 (red trace). Shading around the traces indicates the standard deviation (s.d.) of the corresponding distance. The pre-hydrolytic state is stable and does not undergo any structural rearrangement, but it forms the K1–N7 interaction rapidly after the equilibration phase. b MD simulations of the N7-deaza state (structure of N7-deaza-G in the inset). The absence of the K1–N7 interaction alters the conformation of G288, eventually leading to active site misfolding (dM1–M2 = 5.28 ± 0.12 Å, red trace). c Graphical representation of the K1–N7 interaction modeled from the simulations of the pre-hydrolytic state. The J2/3 junction (purple, ball and stick representation), M1 and M2 (green spheres), and the catalytic triad and two-nucleotide bulge (yellow, ball and stick representation, backbone as ribbon) are highlighted, together with the key descriptors reported in a and b (dotted lines represent dK1-N7G288 and dM1–M2). Disruption of K1–N7 causes G288 and G359 to move from their triple helix conformation (i.e. from the…

• 

  Fig. 5. Protonation of N3C358 favors K1 release. Evolution of dM1–M2 (red trace), dK1–N7 (green trace), and dM2-O6 (blue trace) over the course of MD simulations of: a the cleaved state, and b the cleaved-H+ state. Shading around the traces indicates the s.d. of the corresponding distance Schematic structures of non-protonated and protonated cytidine groups are depicted in the bottom right of each panel. K1 is released after ~300 ns and ~40 ns from the two states, respectively (red arrow). c Structure of the active site at the beginning of the simulations after the formation of K1–N7G288. d Structure of the active site at the end of the simulations, after K1 release. K1 is released after the solvation of the active site (water molecule depicted as a red sphere, W).

• 

  Fig. 6. Energetics associated with intron toggling in the protonated state. a Structural architecture of the active site for the intermediate A, intermediate B, and toggled states identified by the MtD simulation reported in (b). b Path MtD free energy landscape of the cleaved-H+ state. The intermediate A, intermediate B, and toggled states are indicated along the MtD trajectory (dark blue dotted line). The energy scale is indicated in kcal mol−1 on the right. The conformations of the J2/3 junction in state A and in the toggled state are represented at the bottom of the figure.

• 

  Fig. 7. Revised group II intron splicing cycle. a Snapshots of the intron active site derived from the crystal structures and the MD simulations. In the pre-catalytic state, which corresponds to the pre-hydrolytic state of the first step of splicing, a water molecule (light blue sphere, W) or the 2′-OH of a bulged adenosine are poised for nucleophilic attack on the scissile phosphate (orange sphere, SP) (PDB id. 4FAQ). In this state, the intron established the K1–N7G288 interaction, and the nucleophile cleaves the intron-5en junction. Immediately after hydrolytic cleavage, the proton released by the nucleophile initially to the bulk solvent is shuttled to C358 in the catalytic triad. C358 protonation favors release of the metal ions cluster and toggling of J2/3 junction (PDB id. 4FAX). This conformational rearrangement likely prompts the release of the products of the first step of splicing, subsequent rearrangement of D6, and reverse toggling of J2/3 to reconstitute the active site and align second step reactants. Finally, cleavage of intron-3e junction leads to the release of free linear intron (PDB id. 4E8M) and spliced exons. The free intron is still an active ribozyme, which can…