Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Self-cleaving RNAs, like the leadzyme, utilize dynamic conformational changes for catalytic activity.
• Understanding the relationship between RNA dynamics and function is crucial for deciphering biological mechanisms.

Purpose of the Study:

• To investigate the role of conformational dynamics in the self-cleavage function of the lead-dependent ribozyme (leadzyme).
• To elucidate the connection between dynamic fluctuations and chemical activation in ribozymes.

Main Methods:

• Utilized Nuclear Magnetic Resonance (NMR) spin-relaxation analysis to study ribose group dynamics.
• Employed chemical modification with a North-methanocarbacytidine analog to restrict conformational flexibility.
• Assessed self-cleavage activity of both native and conformationally restricted leadzyme variants.

Main Results:

• NMR data revealed conformational exchange at the active site (Cyt-6) of the leadzyme, involving a minor C2'-endo state.
• Conformational restriction, preventing fluctuations to the C2'-endo state, led to a significant decrease in self-cleavage activity.
• The observed dynamics were on the microsecond to tens of microseconds timescale.

Conclusions:

• Dynamic sampling of a minor conformational state is essential for the catalytic self-cleavage function of the leadzyme.
• This finding suggests that RNA dynamics are tightly coupled to catalytic activity in this ribozyme and potentially related motifs.
• The combined approach of NMR dynamics and functional probing offers a general strategy for studying RNA dynamics-function relationships.