Xingwang Fang

Mg2+-dependent folding of a large ribozyme without kinetic traps.

The folding kinetics of the catalytic domain of Bacillus subtilis ribonuclease P is analyzed here by fluorescence and catalytic activity. The folding pathway is apparently free of kinetic traps, as indicated by a decrease in folding rates upon the addition of urea. We apply Mg2+ and urea chevron analysis to fully describe the folding and unfolding kinetics of this ribozyme. A folding scheme containing two kinetic intermediates completely accounts for the free energy, the Mg2+ Hill coefficient and the surface buried in the equilibrium transition. At saturating Mg 2+concentrations, folding is limited by a barrier that is independent of Mg2+ and urea. These results describe the first trap-free folding pathway of a large ribozyme and indicate that kinetic traps are not an obligate feature of RNA folding.

[24] Application of circular dichroism to study RNA folding transitions

Publisher Summary Circular dichroism (CD) spectroscopy has been widely used to study secondary structure formation in both proteins and nucleic acids. In this chapter, the application of CD is used to study RNA folding transitions. Modern commercially available CD spectrometers are capable of simultaneous CD and absorbance measurements. The amplitude of the applied photomultiplier voltage (high tension) directly relates to absorbance of the sample and is recorded along with the CD signal. This enables to measure transitions by both probes without any additional measurements. The chapter also discusses the use of CD in combination with Mg 2+ or urea titrations to study the secondary and tertiary structural transitions in RNAs. Two-domain ribozyme P RNA have been from Bacillus subtilis folds with two distinct structural transitions, an unfolded to intermediate, U-to-I, transition and an intermediate to native transition. The U-to-I transition occurs at micromolar Mg 2+ concentrations and is readily monitored by changes in absorbance (A 260 ) and CD at 260 nm. These signals are primarily sensitive to the formation of helical structure. Tertiary transition occurs in millimolar Mg 2+ range with a Hill coefficient of about 4. The formation of the tertiary structure of RNA is the result of structural changes involving the ribosephosphate backbone and non-Watson–Crick base pairing of nucleotides.

The rate-limiting step in the folding of a large ribozyme without kinetic traps

A fundamental question in RNA folding is the nature of the rate-limiting step. Folding of large RNAs often is trapped by the need to undo misfolded structures, which precludes the study of the other, potentially more interesting aspects in the rate-limiting step, such as conformational search, metal ion binding, and the role of productive intermediates. The catalytic domain of the Bacillus subtilis RNase P RNA folds without a kinetic trap, thereby providing an ideal system to elucidate these steps. We analyzed the folding kinetics by using fluorescence and absorbance spectroscopies, catalytic activity, and synchrotron small-angle x-ray scattering. Folding begins with the rapid formation of early intermediates wherein the majority of conformational search occurs, followed by the slower formation of subsequent intermediates. Before the rate-limiting step, more than 98% of the total structure has formed. The rate-limiting step is a small-scale structural rearrangement involving prebound metal ions.

The thermodynamic origin of the stability of a thermophilic ribozyme

Understanding the mechanism of thermodynamic stability of an RNA structure has significant implications for the function and design of RNA. We investigated the equilibrium folding of a thermophilic ribozyme and its mesophilic homologue by using hydroxyl radical protection, small-angle x-ray scattering, and circular dichroism. Both RNAs require Mg2+ to fold to their native structures that are very similar. The stability is measured as a function of Mg2+ and urea concentrations at different temperatures. The enhanced stability of the thermophilic ribozyme primarily is derived from a tremendous increase in the amount of structure formed in the ultimate folding transition. This increase in structure formation and cooperativity arises because the penultimate and the ultimate folding transitions in the mesophilic ribozyme become linked into a single transition in the folding of the thermophilic ribozyme. Therefore, the starting point, or reference state, for the transition to the native, functional thermophilic ribozyme is significantly less structured. The shift in the reference state, and the resulting increase in folding cooperativity, is likely due to the stabilization of selected native interactions that only form in the ultimate transition. This mechanism of using a less structured intermediate and increased cooperativity to achieve higher functional stability for tertiary RNAs is fundamentally different from that commonly proposed to explain the increased stability of thermophilic proteins.

Folding of a universal ribozyme: the ribonuclease P RNA

Ribonuclease P is among the first ribozymes discovered, and is the only ubiquitously occurring ribozyme besides the ribosome. The bacterial RNase P RNA is catalytically active without its protein subunit and has been studied for over two decades as a model system for RNA catalysis, structure and folding. This review focuses on the thermodynamic, kinetic and structural frameworks derived from the folding studies of bacterial RNase P RNA.

Stepwise Conversion of a Mesophilic to a Thermophilic Ribozyme

A fundamental question in RNA folding is the mechanism of thermodynamic stability. We investigated the equilibrium folding of a series of sequence variants in which one to three motifs of a 255-nucleotide mesophilic ribozyme were substituted with the corresponding motifs from its thermophilic homologue. Substitution of three crucial motifs individually or in groups results in a continual increase in the stability and folding cooperativity in a stepwise fashion. We find an unexpected relationship between stability and folding cooperativity. Without changing the folding cooperativity, RNAs having a similar native structure can only achieve moderate change in stability and likewise, without changing stability, RNAs having a similar native structure can only achieve moderate change in folding cooperativity. This intricate relationship must be included in the predictions of tertiary RNA stability.