Abstract
Rearrangement of RNA secondary structure is crucial for numerous biological processes. RNA helicases participate in these rearrangements through the unwinding of duplex RNA. We report here that the redox-regulated cyanobacterial RNA helicase, CrhR, is a bona fide RNA helicase possessing both RNA-stimulated ATPase and bidirectional ATP-stimulated RNA helicase activity. The processivity of the unwinding reaction appears to be low, because RNA substrates containing duplex regions of 41 bp are not unwound. CrhR also catalyzes the annealing of complementary RNA into intermolecular duplexes. Uniquely and in contrast to other proteins that perform annealing, the CrhR-catalyzed reactions require ATP hydrolysis. Through a combination of the unwinding and annealing activities, CrhR also catalyzes RNA strand exchange resulting in the formation of RNA secondary structures that are too stable to be resolved by helicase activity. RNA strand exchange most probably occurs through the CrhR-dependent formation and resolution of an RNA branch migration structure. Demonstration that another cyanobacterial RNA helicase, CrhC, does not catalyze annealing indicates that this activity is not a general biochemical characteristic of RNA helicases. Biochemically, CrhR resembles RecA and related proteins that catalyze strand exchange and branch migration on DNA substrates, a characteristic that is reflected in the recently reported structural similarities between these proteins. The data indicate the potential for CrhR to catalyze dynamic RNA secondary structure rearrangements through a combination of RNA helicase and annealing activities.
Highlights
The ability of organisms to rearrange nucleic acid secondary structure is crucial for cellular function and is catalyzed by a diverse range of proteins or protein complexes that facilitate nucleic acid annealing and unwinding
We report here that the redox-regulated cyanobacterial RNA helicase, CrhR, is a bona fide RNA helicase possessing both RNA-stimulated ATPase and bidirectional ATP-stimulated RNA helicase activity
ATPase activity was detected in the absence of exogenous RNA, a level that was reduced to background by treatment of E. coli extracts with RNase before affinity purification (Ϫ RNA)
Summary
Expression and Purification of Recombinant CrhR RNA Helicase—An N-terminal translational fusion between a His tag and the CrhR ORF was generated by ligation of two DNA fragments coding for crhR into BamHI/EcoRI-digested pRSET A (Invitrogen). ATPase assays were performed at 37 °C in a 0.4-ml reaction volume containing 20 mM HEPES-KOH, pH 7.5, 80 mM KCl, 3 mM Mg acetate, 1 mM dithiothreitol, 5 mM ATP, 2 mM phosphoenolpyruvate, 0.3 mM NADH, 10 g of poly(A) RNA, 3 units/ml pyruvate kinase and lactate dehydrogenase, and 0.2 g of purified recombinant CrhR. The ssRNA substrates were obtained by heat denaturation of either the 5Ј- or 3ЈdsRNAs for 5 min at 90 °C followed by snap-cooling on ice. Strand exchange assays corresponded to the RNA helicase reactions with the exception that 80 fmol of a third unlabeled 174-nt ssRNA strand, partially complementary to the radiolabeled strand of the 5Ј-tailed dsRNA, was included in the reaction. Anti-CrhR [18] and secondary antibodies (Sigma) were used at dilutions of 1:5000 and 1:20,000, respectively
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