Escherichia coli RNase R Has Dual Activities, Helicase and RNase

When exponentially growing cells of Escherichia coli are shifted from 37°C to a low temperature, such as 15°C, a cold shock response is elicited. This response is characterized by a transient arrest of cell growth termed the acclimation phase, followed by resumption of growth at the low temperature. During the acclimation phase there is severe inhibition of general protein synthesis. However, several cold shock proteins are induced during this phase, including CspA (19) and its homologues, such as CspB (26), CspG (34), and CspI (55), polynucleotide phosphorylase (PNPase) (15, 43, 44), RNA helicase CsdA (49, 50), initiation factor IF2 (20), transcription factor NusA (18), RecA (54), histone-like protein H-NS (14), DNA gyrase (23), and ribosome-binding factor RbfA (12). CsdA is a DEAD-box protein that belongs to the large family of putative RNA helicases. Members of this family are conserved in organisms from bacteria to humans (29) and play important roles in many cellular processes, such as processing, transport, or degradation of RNA or ribosome biogenesis (for a review, see reference 21). CsdA has been identified as a multifunctional protein, and it has been proposed that this protein participates in a variety of processes, such as ribosome biogenesis, mRNA decay, translation initiation, and gene regulation. CsdA is essential at low temperatures, and deletion of its gene impairs growth when there is a cold shock (8, 24). On the other hand, it is dispensable at 37°C. Previously, we showed that the helicase activity of CsdA is pivotal in its role at low temperature (3). Our in vivo genetic screening of an E. coli strain revealed that another DEAD-box RNA helicase, RhlE, can compensate for the CsdA function at low temperature. This finding is also consistent with another report, which showed that RhlE can complement the cold shock function of CsdA and proposed that this protein is involved in ribosome assembly (22). We also observed that although not detected in our genetic screen, an exonuclease, RNase R, can also complement the cold shock function of CsdA (3). RNase R is one of the three major 3′-to-5′ processing exoribonucleases in E. coli; the other two are PNPase and RNase II. These enzymes are involved primarily in RNA metabolism in E. coli. Both PNPase (25, 61) and RNase R (7), but not RNase II, are induced by cold shock. It has been suggested that PNPase and RNase R are the universal degraders of structured RNA in vivo (11, 28). However, RNase R was the only RNase that was able to complement the helicase activity of CsdA (3). This observation led us to hypothesize that complementation of the cold shock function of CsdA by RNase R is not merely due to its ability to degrade secondary structures in RNAs but that it may also possess helicase activity. RNase II and RNase R belong to the RNR family, and their secondary structures exhibit approximately 60% similarity. However, RNase R is larger than RNase II (10). The crystal structures of E. coli RNase II and a catalytic site mutant of it in a complex with single-stranded RNA (ssRNA) have been solved (17, 32, 62). RNase R consists of 813 amino acids. Based on sequence analysis and comparison with the RNase II structure, RNase R contains a central nuclease domain, two cold shock (CSD) domains near the N terminus of the protein, an S1 domain, and a highly basic region near the C terminus of the protein (52). Here we carried out an in vivo domain analysis of RNase R and showed that this protein also has helicase activity and that this activity is essential for complementation of the cold shock function of CsdA. Mutant RNase R proteins lacking the RNase activity were still able to complement the cold shock function of CsdA. On the other hand, the CSD2 domain, which is not essential for the RNase activity of RNase R, was found to be important for its helicase activity. We also showed that the RNase R protein exhibits helicase activity in vitro, which is independent of its RNase activity. Our results shed light on the unique properties of RNase R and how it is distinct from other 3′-to-5′ processing exoribonucleases in E. coli.