A new RNA helicase isolated from HeLa cells that catalytically translocates in the 3' to 5' direction.
We have purified an RNA helicase to near homogeneity from nuclear extracts of HeLa cells. The enzyme migrated as a 130-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and exhibited a sedimentation coefficient of 6.4 on glycerol gradient centrifugation. The enzyme translocated in a 3' to 5' direction and acted catalytically, displacing at least a 4-fold molar excess of duplex RNA compared with the enzyme added. All eight common nucleoside triphosphates supported RNA helicase activity at relatively low concentrations (Km in values in the 15-20 microM level). In the presence of RNA and some single-stranded DNAs, the RNA helicase hydrolyzed all nucleoside triphosphates to nucleoside diphosphates and inorganic phosphate. The enzyme displaced deoxyribooligonucleotides provided they were hydrogen-bonded to RNA possessing 3' single-stranded regions, but it did not displace ribooligonucleotides hydrogen-bonded to DNA containing 3' single-stranded regions. The enzyme, in the absence of ATP, binds to both single-stranded RNA and DNA, but the amount of complex formed with RNA was 20-fold greater than the complex formed with DNA. In both cases, the complex formed in the absence of ATP was rapidly reversed by the addition of ATP and not by adenyl-5'-yl (beta,gamma-methylene)-diphosphate. We propose that the enzyme can bind to both single-stranded RNA and DNA and hydrolyze ATP, but by virtue of its greater stability on RNA, the enzyme can only translocate on RNA possessing 3' single-stranded regions.
Lee
C G
CG
Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Institute, New York, New York 10021.
Hurwitz
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism; D013379:Substrate Specificity
D003854:Deoxyribonucleotides; D009692:Nucleic Acid Heteroduplexes; D012330:RNA, Double-Stranded; D000255:Adenosine Triphosphate; D004247:DNA; D012316:RNA Nucleotidyltransferases; D020365:RNA Helicases