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A general RNA-capping ribozyme retains stereochemistry during cap exchange
1Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, BC, Canada.
Journal of the American Chemical Society
|October 19, 2006
Summary
This study shows an RNA-capping ribozyme retains stereochemistry during reactions, unlike other ribozymes. This suggests a shared evolutionary path for RNA and protein capping catalysts due to chemical necessity.
Area of Science:
- Biochemistry
- Molecular Biology
- RNA Catalysis
Background:
- Ribozymes are RNA molecules with catalytic activity.
- Many known ribozymes invert stereochemistry at the phosphorus center during reactions.
- RNA capping is crucial for mRNA stability and translation.
Purpose of the Study:
- To investigate the stereochemical outcome of an RNA-capping ribozyme reaction.
- To elucidate the kinetic mechanism of RNA capping by this ribozyme.
- To compare the catalytic mechanism with known protein-based capping enzymes.
Main Methods:
- Synthesis of stereochemically defined RNA caps (Rp and Sp isomers).
- Incubation of ribozyme with modified nucleotide substrates and radiolabeled ATP.
- Kinetic analysis of capping, pyrophosphate release, and hydrolysis.
- Stereochemical analysis of the reaction products.
Main Results:
- The RNA-capping ribozyme retains stereochemistry at the phosphorus center, unlike inverting ribozymes.
- Rp-capped substrates reacted, while Sp-capped substrates did not, even with thiophilic ions.
- Capping kinetics followed Michaelis-Menten behavior, with pyrophosphate release independent of substrate concentration.
- The sum of capping and hydrolysis rates equaled pyrophosphate release rate, indicating an early rate-limiting step.
Conclusions:
- The RNA-capping ribozyme mechanism involves two inverting steps separated by a covalent intermediate.
- This mechanism is similar to protein capping enzymes, suggesting convergent evolution driven by chemical constraints.
- Stereochemical retention is a key feature distinguishing this ribozyme's capping mechanism.
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