Characterization of the cold stress-induced cyanobacterial DEAD-box protein CrhC as an RNA helicase

Abstract

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.

Figure 1

Expression and purification of His-tagged CrhC. Histidine-tagged CrhC was overproduced in E.coli and purified by affinity chromatography on a nickel resin column. Lane 1, soluble proteins from uninduced E.coli strain JM109 (pRSET29); lane 2, soluble proteins from cells induced with IPTG (0.5 mM) in the presence of M13 phage/T7 at 20°C for 5 h; lane 3, flow-through collected after incubation of the lysate with Ni–NTA–agarose beads at 4°C for 1 h; lanes 4–6, proteins eluted with imidazole at 20, 20 and 40 mM; lanes 7–9, CrhC containing fractions eluted with 250 mM imidazole. Proteins were resolved by electrophoresis on a 10% SDS–polyacrylamide gel and stained with Coomassie Brilliant Blue. An arrow indicates the position of the 50 kDa His-tagged CrhC. The migration position of molecular weight makers is indicated on the left.

Figure 2

RNA-dependent ATPase activity of CrhC. (A) The effect of RNA concentration on the ATPase activity of CrhC. ATPase reactions were performed with 200 ng recombinant CrhC. The indicated amounts of total RNA isolated from cold-shocked Anabaena were added to the reactions. (B) CrhC ATPase activity is Mg²⁺ dependent. ATPase reactions were performed with 200 ng recombinant CrhC in the presence of 10 µg total RNA isolated from cold-shocked Anabaena. Mg²⁺ was added to the indicated final concentration.

Figure 3

5′→3′ RNA unwinding activity of CrhC. RNA unwinding assays were performed under standard assay conditions (pH 8.5, 200 ng His-tagged CrhC, 50 fmol RNA II dsRNA substrate, 3 mM ATP and 3 mM Mg²⁺ for 10 min at 37°C) and modified as indicated. (A) Structure of the artificial duplex RNA substrate RNA II. (B) Influence of CrhC concentration on RNA unwinding activity. RNA helicase reactions were performed in the presence of the indicated amounts of CrhC. (C) A time course of RNA unwinding. RNA helicase reactions were performed with 20 ng His-tagged CrhC for the indicated times. (D) CrhC RNA unwinding activity is ATP dependent. ATP concentration in the reaction was varied as indicated. (E) CrhC unwinding activity is Mg²⁺ dependent. The Mg²⁺ concentration in the reaction was varied as indicated. (F) RNA unwinding activity of CrhC is ATP dependent. RNA helicase reactions were performed in the presence of the indicated nucleotide triphosphate provided at a final concentration of 3 mM. The percentage of the input duplex substrate RNA II unwound during the reaction is indicated below each lane.

Figure 4

3′→5′ RNA unwinding is not catalyzed by CrhC. (A) Structure of the artificial 3′ tailed duplex RNA substrate. (B) RNA unwinding assays were performed as described for the standard assay in Figure 3 except that the duplex RNA substrate contains only 3′ ssRNA tails. His-tagged CrhC concentration was modified as indicated.

Figure 5

ATP-independent RNA binding activity of CrhC. CrhC binding to RNA II (50 fmol) was assayed in the presence of the indicated amounts of recombinant CrhC, in the presence and absence of ATP (3 mM) at 37°C for 30 min. Reaction products were separated by electrophoresis on native 8% polyacrylamide gels. The migration positions of duplex RNA substrate and RNA–CrhC complexes are indicated.

Figure 6

CrhC is a component of a multisubunit complex at 20°C. Anti-CrhC antiserum was used to immunoprecipitate ³⁵S-labeled polypeptides from lysates prepared from Anabaena cells grown at 20 or 30°C (lanes 1 and 2). Western analysis of immunoprecipitated extracts probed with anti-CrhC antibodies was utilized to identify CrhC in the precipitates (lanes 3 and 4).

Figure 7

CrhC specifically interacts with a constitutively expressed 37 kDa polypeptide. Far-western analysis was utilized to detect polypeptides with which CrhC interacts directly. Immunoblots of soluble protein isolated from Anabaena grown at 20 or 30°C was probed with affinity-purified CrhC (10 µg/ml). Protein–protein interaction was indicated by anti-CrhC antibody binding and colorimetric detection using goat anti-rabbit horseradish peroxidase conjugate (1:1000; Cappel) and 4-chloro-1-napthol. An arrow indicates the presence of CrhC in the 20°C lane.