Characterization of a new RNA helicase from nuclear extracts of HeLa cells which translocates in the 5' to 3' direction.
RNA helicase II, isolated from nuclear extracts of HeLa cells, was purified 1,300-fold and contained a single protein band of 100 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme displaced partial duplex RNA exclusively in a 5' to 3' direction. This reaction was supported only by ATP and deoxy-ATP at relatively high concentrations (the Km was estimated as 1 mM). The enzyme displayed only ATPase and deoxy-ATPase activity that was stimulated preferentially by poly(C). RNA helicase catalyzed the unwinding of duplex RNA and RNA.DNA hybrids provided that single-stranded (ss) RNA was available for the helicase to bind. In the presence of MgCl2 and ATP or adenosine 5'-O-(3-thiotriphosphate) RNA helicase II interacted with ssRNA and yielded a protein-RNA complex only when reaction mixtures were treated with glutaraldehyde after incubation. When the reactions contained nonhydrolyzable ATP analogs or GTP, ssRNA was converted into an electrophoretically slower migrating form by the helicase. The slower migrating RNA form was shown to be an RNA species containing secondary structure that resided within a putative hairpin loop. These observations indicate that RNA helicase II can introduce intramolecular secondary structure in ssRNA.
Flores-Rozas
H
H
Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Institute, New York, New York 10021.
Hurwitz
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D015636:Magnesium Chloride / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics
D012333:RNA, Messenger; D015636:Magnesium Chloride; D012316:RNA Nucleotidyltransferases; D000251:Adenosine Triphosphatases; D020365:RNA Helicases