PMC

A schematic of the D1356 ribozyme construct derived from the ai5γ group II intron.

(a) Comparison of global compaction of the D1356 ribozyme folded at various magnesium concentrations at 42 °C (left) with that after folding to the native state at 42 °C and 100 mM MgCl₂ followed by dilution to the same magnesium concentrations at 30 °C (right). (b) Comparison of global compaction of the Tetrahymena group I intron upon folding at various magnesium concentrations (left) with that after folding to the native state and dilution to the same magnesium concentrations (right).

(a) A scheme illustrating folding pathway of the ai5γ-derived ribozyme at 30 °C. (b) A scheme of a hypothetical free energy diagram for the ribozyme folding to the near-native state and native states at low magnesium (solid black lines). Hypothetical changes in free energy landscape upon adding high concentrations of magnesium or protein co-factors are shown in red.

Comparison of global compaction of the D1356 ribozyme at different magnesium concentrations after 20 minute incubation at 42 and 30 °C. ME, sample incubated in 10 mM MOPS, pH 6.0, 1 mM EDTA at 95 °C for 1 min and used as a control for the mobility of unfolded species.

Unfolding of the compact intermediate in the presence of increasing concentrations of urea. Representative native gels showing urea-induced unfolding of the intermediate, which was either fully compacted at 30 °C and 5 mM MgCl₂ (a) or diluted to the same concentration of magnesium from the native state (c). The fraction of compact population was plotted vs urea concentration and analyzed as previously described in order to determine free energy of unfolding at different magnesium concentrations for compact species formed either after slow compaction at 30 °C (b) or after dilution from the native state (d).

Multiple turnover kinetic analysis of RNA oligonucleotide substrate cleavage by the D1356 ribozyme. The ribozyme was either preincubated to full compaction at 30 °C and 3, 5, 10 or 20 mM MgCl₂ ((a), (b)) or prefolded to a native state at 100 mM MgCl₂ and 42 °C and then diluted to the same magnesium concentrations as above ((c), (d)). All reactions were carried out at 30 °C and either 3, 5, 10 or 20 mM MgCl₂, respectively, ((a), (c)) or at 100 mM MgCl₂ ((b), (d)). Unfolded and native ribozyme samples were used as controls for comparison of rate constants ((b), (d)).

A time course of the D1356 ribozyme compaction at 30 °C. Native gel electrophoresis was used to monitor compaction over time (a). The fraction of compact population was plotted vs time and fit to a first order kinetic equation to calculate rate constants for compaction at different magnesium concentrations (b). (c) Comparison of the compaction time courses in the presence and in the absence of subdenaturing concentrations of urea at 5 mM and 100 mM MgCl₂. The rate constants for compaction at 5 mM MgCl₂ were 0.004±0.001 min⁻¹ and 0.005±0.002 min⁻¹ in the absence and presence of 0.5 M urea, respectively. The rate constants for compaction at 100 mM MgCl₂ were 0.033±0.002 min⁻¹ and 0.032±0.001 min⁻¹ in the absence and presence of 1 M urea, respectively.

DMS structural probing of the compact folding intermediate. (a) Representative sequencing gels showing A and C positions protected against DMS modification already at 3 mM MgCl₂ (red arrows). Prior to DMS treatment, samples were incubated at indicated magnesium concentrations at 30 °C until fully compact. (b) Representative sequencing gels showing A and C residues that gradually become protected against DMS at magnesium concentrations higher than 3 mM (green arrows). Yellow arrows indicate residues that are partially protected at 3 mM MgCl₂ and gradually become fully protected at higher magnesium concentrations. Samples were treated as above (see legend to ). (c) The structure of the intermediate formed after dilution from the native state to indicated magnesium concentrations is identical to that of the intermediate formed by slow collapse at the same magnesium concentrations (compare to ). Representative sequencing gels show A and C positions protected from DMS already at 3 mM MgCl₂ (red arrows), gradually becoming protected with increasing magnesium (green arrows) or partially protected at 3 mM MgCl₂ and gradually becoming fully protected at higher concentrations of magnesium (yellow arrows). (d) A bar graph showing extent of protection (%) at different magnesium concentration relative to the native state (100 %) for representative A and C positions that are fully protected already at 3 mM MgCl₂ (shown in black and grey) and regions in D1 (blue), D5 (red) and D3 (green) that gradually become protected upon increasing magnesium. (e) The secondary structure map of the D1356 ribozyme showing regions corresponding to the compact intermediate (near-native state) (red) and regions gradually forming during the transition from the near-native to native state (green). Regions that are partially protected in the near-native state and gradually become fully protected in the native state are shown in yellow.