Characterization of the cold stress-induced cyanobacterial DEAD-box protein CrhC as an RNA helicase.
We have shown previously that CrhC is a unique member of the DEAD-box family of RNA helicases whose expression occurs specifically under conditions of cold stress. Here we show that recombinant His-tagged CrhC, purified from Escherichia coli, is an ATP-independent RNA binding protein possessing RNA-dependent ATPase activity which is stimulated most efficiently by rRNA and polysome preparations. RNA strand displacement assays indicate that CrhC possesses RNA unwinding activity that is adenosine nucleotide specific. Unwinding of partially duplexed RNA proceeds in the 5'-->3' but not the 3'-->5' direction using standard assay conditions. Immunoprecipitation and far-western analysis indicate that CrhC is a component of a multisubunit complex, interacting specifically with a 37 kDa polypeptide. We propose that CrhC unwinds cold-stabilized secondary structure in the 5'-UTR of RNA during cold stress.
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Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
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D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D017033:Anabaena / Q000201:enzymology*; D002384:Catalysis; D002412:Cations / Q000378:metabolism; D002846:Chromatography, Affinity; D003067:Coenzymes / Q000378:metabolism; D003080:Cold Temperature; D046911:Macromolecular Substances; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D020365:RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D002412:Cations; D003067:Coenzymes; D046911:Macromolecular Substances; D010455:Peptides; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012313:RNA; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D020365:RNA Helicases