Kinetic scheme for intermolecular RNA cleavage by a ribozyme derived from hepatitis delta virus RNA

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Catalysis

Background:

• Trans-acting ribozymes, derived from self-cleaving RNA elements like the HDV antigenomic RNA, play crucial roles in RNA metabolism.
• Understanding the detailed kinetic mechanisms of these ribozymes is essential for elucidating their catalytic functions and potential applications.

Purpose of the Study:

• To establish a minimal kinetic mechanism for a trans-acting ribozyme from the HDV antigenomic RNA.
• To determine rate constants for individual steps, including substrate binding, cleavage, and product release.

Main Methods:

• Utilized steady-state, pre-steady-state, single-turnover, and binding kinetics.
• Measured rate constants at 37°C, pH 8.0, in 10 mM Mg(2+) using synthetic oligonucleotides.
• Employed substrates, noncleavable analogs, and 3' product mimics.

Main Results:

• The 3',5'-linkage substrate cleavage rate constant (k2) was 0.91 min⁻¹.
• Substrate dissociation (1.4 min⁻¹) was faster than cleavage, indicating the binary complex was not at equilibrium.
• Product release was ordered: 5' product released rapidly (>12 min⁻¹), while 3' product released slowly (6.0 x 10⁻³ min⁻¹), making it rate-limiting for turnover.
• Cleavage of a 2',5'-linkage substrate followed Michaelis-Menten kinetics, distinct from the Briggs-Haldane mechanism for 3',5'-linkage cleavage.

Conclusions:

• The slow release of the 3' product limits the overall catalytic turnover rate.
• The rapid release of the 5' product and lack of a binding site may hinder ribozyme-catalyzed ligation (reverse reaction).
• The kinetic mechanism differs based on the linkage type at the cleavage site (3',5' vs. 2',5').