Related Experiment Videos
Action of Pennisetum typhoides double-stranded ribonuclease on viral ds RNAs
Abstract:
A ribonuclease that preferentially cleaves natural and synthetic double-stranded (ds) RNAs has been partially purified from Pennisetum typhoides. The enzyme degrades [3H]poly(rA) X poly(rU) to acid-soluble products. The ds RNase does not degrade RNA:DNA hybrid but appears to have about 18% activity against single-stranded (ss) RNAs under the assay conditions used for the cleavage of ds RNAs. The RNase has a molecular weight of 35,000 daltons as determined from gel filtration using Sephadex G-200. The ds RNase shows an absolute requirement for divalent cations Mg++ or Mn++ and monovalent cations K+ or Na+. The specificity of the enzyme towards ds RNA template is supported by the inhibition of cleavage of ds RNAs by ethidium bromide and Penicillium chrysogenum viral ds RNA. The enzyme preparation acts on ds RNAs isolated from Saccharomyces cerevisiae and P. chrysogenum virus. The purified ds RNase also cleaves the in vitro transcriptional products of adenovirus DNA and this activity is inhibited by 5 mM ethidium bromide suggesting that ds regions of adenovirus RNA are involved in the cleavage process.
Insights
Researchers identified a double-stranded RNAse from Pennisetum typhoides that specifically degrades double-stranded (ds) RNA. This enzyme requires specific cations and shows selectivity for ds RNA over single-stranded RNA or RNA:DNA hybrids.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Double-stranded ribonucleic acid (dsRNA) plays critical roles in various biological processes, including viral replication and gene regulation.
- Understanding enzymes that interact with dsRNA is crucial for deciphering these roles and developing therapeutic strategies.
Purpose of the Study:
- To partially purify and characterize a novel ribonuclease (RNase) from Pennisetum typhoides with preferential activity against double-stranded RNAs.
- To investigate the substrate specificity, optimal conditions, and molecular properties of the purified dsRNA-specific RNase.
Main Methods:
- Partial purification of the dsRNA-specific ribonuclease from Pennisetum typhoides using biochemical techniques.
- Enzyme activity assays using synthetic and natural dsRNA substrates, including [3H]poly(rA) x poly(rU).
- Determination of molecular weight via gel filtration (Sephadex G-200).
- Assessment of cofactor requirements (divalent and monovalent cations) and substrate specificity (dsRNA, ssRNA, RNA:DNA hybrids).
Main Results:
- A ribonuclease preferentially cleaving natural and synthetic dsRNAs was partially purified.
- The enzyme degraded [3H]poly(rA) x poly(rU) and showed minimal activity against RNA:DNA hybrids and single-stranded RNAs (ssRNAs).
- The enzyme has a molecular weight of 35,000 daltons and requires Mg++ or Mn++ and K+ or Na+ for activity.
- Inhibition by ethidium bromide and viral dsRNA confirmed specificity for dsRNA templates, with activity observed on viral RNAs and adenovirus RNA transcripts.
Conclusions:
- A novel dsRNA-specific ribonuclease from Pennisetum typhoides has been identified and partially characterized.
- The enzyme exhibits strict substrate specificity for dsRNA, requiring specific cation cofactors.
- This dsRNase has potential applications in studying viral RNA and gene expression involving dsRNA intermediates.