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Double-stranded RNA: the variables controlling its degradation by RNases
G I Yakovlev1, S Sorrentino, G P Moiseyev
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
Nucleic Acids Symposium Series
|January 1, 1995
Summary
This study investigated how three mammalian ribonucleases cleave single-stranded and double-stranded RNA. Enzyme surface charge and salt concentration influenced RNA cleavage activity, particularly for seminal ribonucleases.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Mammalian pancreatic ribonucleases (RNases) are crucial enzymes involved in RNA metabolism.
- Understanding RNase kinetics and substrate specificity is vital for molecular biology applications.
- Previous studies have focused on RNase A, but less is known about seminal RNases' substrate preferences.
Purpose of the Study:
- To compare the cleavage kinetics of single-stranded (ss) and double-stranded (ds) polyribonucleotides by bovine RNase A, bovine seminal RNase, and human seminal RNase.
- To investigate the influence of low and high salt conditions on RNase activity.
- To elucidate the mechanisms underlying RNase activity towards dsRNA substrates.
Main Methods:
- Enzymatic assays were performed to determine kinetic parameters (kcat, Km, kcat/Km) for RNase-mediated depolymerization of various ssRNA and dsRNA substrates (poly(U), poly(A).poly(U), poly(I), poly(I).poly(C)).
- Experiments were conducted under both low and high ionic strength (salt) conditions.
- Enzyme surface charge distribution was correlated with observed activity differences.
Main Results:
- Bovine RNase A exhibited similar Km values for ssRNA and dsRNA substrates under varying salt conditions, but significantly different kcat values.
- Bovine and human seminal RNases showed ssRNA/dsRNA activity ratios primarily dictated by kcat ratios.
- Increased positive surface charges on seminal RNases correlated with higher dsRNA activity, especially under low salt conditions, suggesting a mechanism involving transiently exposed ssRNA sequences.
Conclusions:
- RNase activity towards dsRNA can occur via binding to transiently unwound single nucleotides or exposed ssRNA sequences.
- Seminal RNases, with higher positive surface charges, demonstrate enhanced dsRNA cleavage, particularly at low ionic strength.
- The study provides insights into the substrate specificity and catalytic mechanisms of mammalian pancreatic-type ribonucleases.