Double-stranded RNA: the variables controlling its degradation by RNases.
The kinetics of single-stranded (SS) and double-stranded (ds) polyribonucleotides cleavage by three mammalian pancreatic type ribonucleases have been studied under low and high salt conditions. The values kcat, Km, and kcat/Km for depolymerization of poly(U), poly(A).poly(U), poly(I) and poly(I).poly(C) by bovine RNase A, bovine seminal RNase, and human seminal RNase have been determined and compared to each other. The Km values of bovine RNase A for (ss) or (ds) substrates were of the same order of magnitude under low and high ionic strength conditions, while their kcat values were found to differ considerably. Qualitatively similar results were obtained with bovine and human seminal RNases, i.e., the activity ratios (ssRNA/dsRNA) were mostly determined by the ratio of kcat values. It was shown that the modest levels of activity toward dsRNAs shown by single-strand-preferring RNases may occur by a mechanism consisting in the binding of the RNase to single nucleotides which are wound off the double helix because of thermal fluctuations. A higher activity and its enhancement as a function of number and location of the positive charges present on the RNase surface (human seminal RNase > bovine seminal RNase > bovine RNase A), as well as its increase under low ionic strength conditions, could instead be explained by the increased occurence of the splitting mechanism based on the binding of the RNase to single-stranded RNA sequences transiently exposed from the RNA double-helix.
Yakovlev
G I
GI
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
Sorrentino
S
S
Moiseyev
G P
GP
Libonati
M
M
D000818:Animals; D002417:Cattle; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D009994:Osmolar Concentration; D010179:Pancreas / Q000201:enzymology; D011063:Poly A-U / Q000378:metabolism; D011069:Poly I / Q000378:metabolism; D011070:Poly I-C / Q000378:metabolism; D011072:Poly U / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*; D012661:Semen / Q000201:enzymology; D013379:Substrate Specificity
D012330:RNA, Double-Stranded; D011063:Poly A-U; D011069:Poly I; D011072:Poly U; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D011070:Poly I-C