Escherichia coli RNase R has dual activities, helicase and RNase

Abstract

In Escherichia coli, the cold shock response occurs when there is a temperature downshift from 37 degrees C to 15 degrees C, and this response is characterized by induction of several cold shock proteins, including the DEAD-box helicase CsdA, during the acclimation phase. CsdA is involved in a variety of cellular processes. Our previous studies showed that the helicase activity of CsdA is critical for its function in cold shock acclimation of cells and that the only proteins that were able to complement its function were another helicase, RhlE, an RNA chaperone, CspA, and a cold-inducible exoribonuclease, RNase R. Interestingly, other major 3'-to-5' processing exoribonucleases of E. coli, such as polynucleotide phosphorylase and RNase II, cannot complement the cold shock function of CsdA. Here we carried out a domain analysis of RNase R and showed that this protein has two distinct activities, RNase and helicase, which are independent of each other and are due to different domains. Mutant RNase R proteins that lack the RNase activity but exhibit the helicase activity were able to complement the cold shock function of CsdA, suggesting that only the helicase activity of RNase R is essential for complementation of the cold shock function of CsdA. We also observed that in vivo deletion of the two cold shock domains resulted in a loss of the ability of RNase R to complement the cold shock function of CsdA. We further demonstrated that RNase R exhibits helicase activity in vitro independent of its RNase activity. Our results shed light on the unique properties of RNase R and how it is distinct from other exoribonucleases in E. coli.

FIG. 1.

Comparison of ribosome profiles of ΔcsdA and ΔcsdA Δrnr cells. ΔcsdA and ΔcsdA Δrnr cells were grown in LB medium with kanamycin at 37, 30, 25, and 20°C until the OD₆₀₀ was 0.5. Polysomes were isolated and resolved as described in Materials and Methods.

FIG. 2.

Domain analysis of RNase R with respect to its ability to complement the cold shock function of CsdA. (A) Schematic diagram of domains of RNase R and plasmids that were constructed. Different domains of RNase R were cloned in the pINIII vector using NdeI and BamHI sites, as indicated. (B) E. coli ΔcsdA Δrnr cells were transformed with plasmid pINIII alone or plasmid pINIII containing CsdA, RNase R, or different domains of RNase R. The plasmids carrying different domains of RNase R are designated as shown in panel A. The cells were streaked on LB medium plates containing ampicillin (50 μg ml⁻¹) and incubated at 37°C, 20°C, and 15°C for the times indicated.

FIG. 3.

RNase activity of RNase R is not required for the ability to complement the helicase activity of CsdA that is essential for its cold shock function. E. coli wild-type cells were transformed with plasmid pINIII as a control, and ΔcsdA cells were transformed with plasmid pINIII alone as a control or plasmid pINIII expressing CsdA, RNase R, or RNase R having mutations in the RNA catalytic domains, as indicated. The cells were streaked on LB medium plates containing ampicillin (50 μg ml⁻¹). The results for plates incubated at 37°C for 24 h and at 15°C for 120 h are shown.

FIG. 4.

RNase R exhibits helicase activity in vitro. (A) Helicase assay carried out at 37°C as described in Materials and Methods using substrate 1 (pds10-3′U₂₀). The products were analyzed by native PAGE followed by phosphorimaging analysis. Lanes SS, heat-denatured substrate showing ssRNA; lanes −P, control reaction without added protein; CsdA-ΔC, reactions carried out with CsdA-ΔC; RNaseR-WT, reactions carried out with wild-type RNase R; D272NRNase R, reactions carried out with D272N RNase R; D278NRNase R, reactions carried out with D278N RNase R; D280NRNase R, reactions carried out with D280N RNase R; lanes 0, 1, 3, 5, 10, 15, and 20, reaction mixtures incubated for 0, 1, 3, 5, 10, 15, and 20 min, respectively. The position of the single-stranded RNA is indicated by an arrow. The labeled strand is indicated by an asterisk. The experiment was repeated two times. (B) Results of a quantitative analysis of the helicase activity of the CsdA-ΔC and RNase R proteins corresponding to the gel shown in panel A.

FIG. 5.

In vitro helicase activity of RNase R with an 18-bp RNA substrate. (A) In vitro helicase assay carried out as described in Materials and Methods using substrate 2 (pds18-3′U₂₀).The products were analyzed by native PAGE followed by phosphorimaging analysis. Lanes SS, heat-denatured substrate showing ssRNA; lanes −P, control reaction without added protein; CsdA-ΔC, reactions carried out with CsdA-ΔC; RNaseR-WT, reactions carried out with wild-type RNase R; D272NRNase R, reactions carried out with D272N RNase R; D278NRNase R, reactions carried out with D278N RNase R; D280NRNase R, reactions carried out with D280N RNase R; lanes 0, 1, 3, 5, 10, 15, and 20, reaction mixtures incubated for 0, 1, 3, 5, 10, 15, and 20 min, respectively. The position of the single-stranded RNA is indicated by an arrow. Labeled strands are indicated by an asterisk. (B) Results of a quantitative analysis of the helicase activity of the CsdA-ΔC and RNase R proteins corresponding to the gel shown in panel A. The experiment was repeated three times. (C) Assay carried out like the assay described above for panel A, but without proteins. The position of ssRNA is indicated. The labeled strands is indicated by an asterisk.