(a) Secondary structure of Osa 1-4 Twister ribozyme. Pseudoknots 1 and 2 are blue and orange, respectively. The arrowhead represents the cleavage site. A P3 stem (faded red letters) was added to Osa 1-4 to stabilize base pairing between substrate (S) and ribozyme (R) strands of bi-molecular ribozymes for activity assays. (b) Structure of Osa 1-4 Twister ribozyme (pdb: 4OJI) colored as in a. Mg²⁺ ions are shown as magenta spheres. (c) Representative 16% PAGE image showing the consumption of substrate (S) and appearance of product (5′P) with time. (d) Progress curves were fitted to single or double exponential rate equations: ƒ_cleaved = I_5′P/(I_S+I_5′P) = A_fast[1–exp(-k_fastt)] + A_slow[1–exp(-k_slowt]. The rate constants are listed in (n = 3; s.d. <10%). (e) Cleavage reactions of the bimolecular riboyzme in different MgCl₂ concentrations were stopped after 1 min and analyzed by denaturing 16% PAGE. (f) The fraction cleaved after 1 min versus Mg²⁺ concentration (filled symbols) was fit to the Hill equation (solid line). n = 3; s.d. < 10%. Relative folding of the bimolecular ribozyme by RNase T1 protection of G21 with MgCl₂ concentration (open symbols; ) was fit to the Hill equation (dashed line) with a midpoint of 2.5 mM MgCl₂. (g) Self-cleavage of the unimolecular ribozyme after 1 min in different MgCl₂ concentrations, as in d. (h) The fraction cleaved (filled symbols and solid line) and RNase T1 protection (open symbols and dashed line; see ) were fit to the Hill equation as in f; n = 3.

(a) The FRET assay for folding. Cy5 is attached to a DNA strand hybridized to a 3′ extension of the ribozyme; Cy3 is attached to the phosphodiester backbone between G23 and U24. In the unfolded state, Cy3 and Cy5 dyes are far apart, resulting in low FRET (left). In the presence of Mg²⁺, pseudoknot formation brings Cy3 and Cy5 together, resulting in high FRET (right). Self-cleavage is prevented by a deoxyribose at the cleavage site. (b) Relative fraction of folded RNA (See for parameters and error analysis) as a function of MgCl₂ concentration, from ensemble FRET at 30 ˚C (filled symbols; left axis) or smFRET at 20 ˚C (open symbols; right axis). (c) smFRET traces at different MgCl₂ concentrations show fluctuations between the low-FRET unfolded state and high-FRET folded state. Blue, Cy3 intensity; red, Cy5 intensity; black, FRET efficiency. With increasing MgCl₂, each molecule spends a longer time in the high FRET state. (d) FRET histograms of Twister ribozyme at various MgCl₂ concentrations showing that total occupancy of the high-FRET state increases with MgCl₂. The histograms were fit to a double Gaussian (black line).

(a) smFRET traces of WT, G21A, G36A and G35A-G36A Twister ribozymes at 50 mM MgCl₂. Twister mutants visited the high FRET folded state infrequently. (b) FRET histograms as in .

(a) A representative experimental FRET trace (black) with idealized FRET trace (red) generated by the HaMMy algorithm. (b) Relaxation rate constants for folding (black) and unfolding (red) versus Mg²⁺ ion concentration at room temperature. The rate constants were fit to a binding model with Mg²⁺ midpoints of saturation 21 (±9) mM and <1 (±0.1) mM for k_F and k_U, respectively. (c) Schematic of the single-molecule folding kinetics experiment. Imaging buffer plus 10 mM MgCl₂ and 5 nM Cy3 dye was added to the slide chamber containing immobilized Cy3-Twister•Cy5-SA5 complexes while recording continuously. The increase in background from free Cy3 marked the diffusion of Mg²⁺-containing buffer. (d) Probability density maps of synchronized Twister FRET dynamics. The red dotted line indicates the moment Mg²⁺ ions interacted with the Twister complex. Before the addition of Mg²⁺, all the molecules were in the 0.2 FRET state. After interacting with Mg²⁺, molecules start fluctuating between the 0.2 and 0.8 FRET states before reaching equilibrium after 5 s.

(a) Twister ribozyme activity after 1 min in MgCl₂, MnCl₂, and ZnCl₂ as in . The midpoints of catalysis for MgCl₂, MnCl₂, and ZnCl₂ were 135 (± 12) μM, 59 (± 5) μM and 51 (± 6) μM, respectively. Values are the mean and s.d. of 2–4 independent trials. (b) Folding in the presence of the divalent ions shown in the key by ensemble FRET at 30 ˚C. The fraction folded was fit to a two-state model as shown in . The folding midpoints were 10 (± 1) mM MgCl₂, 1.7 (± 0.4) mM MnCl₂ and 1.4 (± 0.3) mM ZnCl₂ (± s.d.; n = 2). (c) smFRET traces and (d) FRET histograms at 2 mM MgCl₂, 2 mM MnCl₂ and 2 mM ZnCl₂. Colors are as in . The high-FRET population follows the trend Mg²⁺ < Mn²⁺ < Zn²⁺, suggesting that Twister folds more efficiently in the presence of transition metal ions. (e) Co-ordination of a hydrated Mg²⁺ ion between U-1, A34 and G35. Transition metal ions are expected to directly coordinate G N7 and interact with the phosphophodiester indirectly through a water molecule. (f) Midpoints of self-cleavage in the presence of different divalent salts were determined as described in a and summarized as a horizontal bar graph. Error bars are s.d. of 2–3 independent trials.