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Double-stranded RNA specific nuclease from germinating embryos of Pennisetum typhoides
Abstract:
A double-stranded RNA specific nuclease (ds RNase) has been purified from the pearl millet Pennisetum typhoides. The purification involved S-30 preparation from the germinating embryos, DEAE-cellulose and DNA-cellulose chromatography. The partially pure enzyme preferentially solubilized the synthetic double-stranded polynucleotide [3H]poly(rA) . poly(rU); the degradation of [3H]poly(rC) was fourteen fold lower under the same assay conditions. Furthermore, the ds RNase activity was inhibited to an extent of 58% by ethidium bromide, which is known to intercalate with double-stranded RNAs. Active sulfhydryl groups were found to be necessary for the ds RNase activity since the enzyme action was inhibited by N-ethylmaleimide. Ethidium bromide and N-ethyl-maleimide did not significantly inhibit the ss RNase activity. In contrast, diethyl pyrocarbonate inhibited ss RNase activity completely and ds RNase by 58%. Heating the enzyme for 20 min at 50 degrees C resulted in drastic loss of both enzyme activities. The ds RNase showed maximum activity in the pH range of 6.5 to 7.5. The enzyme acts in vitro on E. coli 30S precursor ribosomal RNA and the cleavage products migrated in the region of mature 23S and 16S rRNAs.
Insights
Pearl millet yields a novel double-stranded RNA specific nuclease (ds RNase). This enzyme preferentially degrades double-stranded RNA and requires active sulfhydryl groups for activity, offering insights into RNA processing.
Area of Science:
- Molecular Biology
- Enzymology
- Plant Biochemistry
Background:
- Double-stranded RNA specific nucleases (ds RNases) play crucial roles in RNA metabolism and defense.
- Understanding the properties of plant-derived ds RNases is essential for exploring their biological functions.
- Pearl millet (Pennisetum typhoides) is a significant cereal crop with potential for novel enzyme discovery.
Purpose of the Study:
- To purify and characterize a double-stranded RNA specific nuclease (ds RNase) from pearl millet.
- To investigate the substrate specificity and optimal conditions for the purified ds RNase.
- To determine the enzyme's sensitivity to various inhibitors and its activity on precursor ribosomal RNA.
Main Methods:
- Purification of ds RNase from pearl millet germinating embryos using S-30 preparation and ion-exchange chromatography (DEAE-cellulose and DNA-cellulose).
- Enzyme activity assays using synthetic polynucleotides ([3H]poly(rA) . poly(rU) and [3H]poly(rC)) and analysis of inhibition by ethidium bromide, N-ethylmaleimide, and diethyl pyrocarbonate.
- In vitro activity assessment on E. coli 30S precursor ribosomal RNA and analysis of cleavage products via gel electrophoresis.
Main Results:
- A partially purified ds RNase was obtained, showing preferential degradation of double-stranded RNA over single-stranded RNA.
- Enzyme activity was inhibited by ethidium bromide and N-ethylmaleimide, indicating the necessity of active sulfhydryl groups and intercalation with ds RNA.
- The ds RNase exhibited optimal activity at pH 6.5-7.5 and cleaved E. coli 30S precursor rRNA into fragments resembling mature 23S and 16S rRNAs.
Conclusions:
- Pearl millet contains a ds RNase with specific activity against double-stranded RNA structures.
- The enzyme's properties suggest a role in RNA processing or regulation, with conserved requirements for activity.
- This purified ds RNase provides a valuable tool for studying RNA structure and function in vitro.