Abstract
An RNA helicase, isolated from nuclear extracts of HeLa cells, displaced duplex RNA in the presence of any one of the eight common nucleoside triphosphates. The unwinding reaction was supported most efficiently by ATP and GTP and poorly by dCTP and dTTP. The enzyme activity, purified 300-fold, contained two major protein bands of 80 and 55 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All fractions that contained RNA helicase activity also possessed single-stranded RNA-dependent nucleoside triphosphatase activity. Purified RNA helicase fractions displaced a hybrid of U4/U6 RNAs with the same efficiency as it displaced other duplex RNA structures. In contrast, the RNA helicase did not displace duplex RNA/DNA and DNA/DNA structures. Evidence is presented that suggests that this RNA helicase can displace duplex RNA by translocating in both the 3' to 5' and the 5' to 3' directions. The properties of the RNA helicase described here differ from the deaminase RNA unwinding activity described in Xenopus oocytes (Bass, B.L., and Weintraub, H. (1987) Cell 48, 607-613) and from the p68 HeLa RNA helicase (Hirling, H., Scheffner, M., Restle, T., and Stahl, H. (1989) Nature 339, 562-564).
Highlights
From the $Program in MolecularBiology, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Institute, New York, New York 10021 and the §Division of Biology 147-75, California Institute of Technology, Pasadena, Californ9ia1125
Purified RNA helicase fractions displaced a hybrid of U4/U6 RNAs with the same efficiency asit displaced other duplex RNA structures
The resultspresented here describe the isolation of an Reactions were incubated a t 30 "C for 20 min, and aliquots were electrophoresed on a 14% acrylamide gel
Summary
The yeast clones of U4and U6 small nuclear RNAs were a generous gift of Drs P. The pellet was suspended in a solution (40 ml) containing 40% ammonium sulfate in buffer. The labeled oligonucleotides were purified by gel electrophoresis and annealed to 4x174 circular ssDNA as described above, except that thehybridization was carried out a t 45 "C (40 ml) containing 30% ammonium sulfate in buffer A plus 0.1 M KC1 and centrifuged; the pellet was re-extracted with the same buffer and again centrifuged. The supernatants obtained after washing with 20% ammonium sulfate helicase was carried out in reaction mixtures (20 pl) containing 40 were pooledand precipitated by addition of solid ammonium mM Tris-HC1 (pH 7.5), 1mM MgC12,3 mM ATP, 2 mM DTT, 0.15 M sulfate to 30%saturation.
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