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Mixed DNA/RNA polymers are cleaved by the hammerhead ribozyme
J H Yang1, J P Perreault, D Labuda
1Département de biochimie, Université de Montréal, Québec, Canada.
Biochemistry
|December 25, 1990
Summary
Hammerhead ribozymes can cleave DNA/RNA mixed polymers, but these substrates bind less effectively. This impaired binding affects the ribozyme-substrate complex conformation and catalytic efficiency.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Hammerhead ribozymes are catalytic RNA molecules known for self-cleavage.
- Substrate specificity and binding are crucial for ribozyme activity.
- Understanding how modified substrates affect ribozyme function is key to exploring their catalytic mechanisms.
Purpose of the Study:
- To investigate the cleavage of DNA/RNA mixed polymers by the hammerhead ribozyme.
- To characterize the kinetic and binding properties of these modified substrates.
- To elucidate the impact of deoxyribonucleotides on ribozyme-substrate complex formation and conformation.
Main Methods:
- Chemical synthesis of oligodeoxyribonucleotides with embedded ribonucleotides.
- Kinetic analysis of ribozyme-mediated cleavage at different temperatures.
- Determination of binding affinities using inhibition assays and thermal denaturation (Tm).
Main Results:
- DNA/RNA mixed polymers are cleaved by hammerhead ribozymes, albeit with altered kinetics.
- Predominantly deoxyribonucleotide substrates exhibit lower optimal cleavage temperatures, higher Km, and lower kcat values.
- Impaired binding of deoxyribonucleotide substrates was observed, confirmed by inhibition and Tm studies.
Conclusions:
- The hammerhead ribozyme can process substrates containing deoxyribonucleotides.
- Incorporation of deoxyribonucleotides into the substrate impairs ribozyme binding and alters catalytic efficiency.
- The presence of DNA/RNA heteroduplexes in the complex induces conformational changes in the ribozyme's catalytic core.