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Background: Escherichia coli RNase R contains an intrinsic RNA helicase activity. Results: Walker A and Walker B motifs located in the C-and N-terminal regions, respectively, are required for helicase activity. Conclusion: RNase R helicase activity utilizes the nuclease catalytic channel and stimulates the nuclease activity. Significance: These findings help to explain how RNase R efficiently degrades structured RNA.

RNase R, a ubiquitous 3 exoribonuclease, plays an important role in many aspects of RNA metabolism. In contrast to other exoribonucleases, RNase R can efficiently degrade highly structured RNAs, but the mechanism by which this is accomplished has remained elusive. It is known that RNase R contains an unusual, intrinsic RNA helicase activity that facilitates degradation of duplex RNA, but how it stimulates the nuclease activity has also been unclear. Here, we have made use of specifically designed substrates to compare the nuclease and helicase activities of RNase R. We have also identified and mutated several residues in the S1 RNA-binding domain that are important for interacting with duplex RNA and have measured intrinsic tryptophan fluorescence to analyze the conformational changes that occur upon binding of structured RNA. Using these approaches, we have determined the relation of the RNA helicase, ATP binding, and nuclease activities of RNase R. This information has been combined with a structural analysis of RNase R, based on its homology to RNase II, whose structure has been determined, to develop a detailed model that explains how RNase R digests structured RNA and how this differs from its action on singlestranded RNA.RNase R is a ubiquitous 3Ј to 5Ј exoribonuclease that, by itself, is able to digest extensively structured RNA molecules (1-4). In vivo, RNase R is primarily a degradative enzyme that acts on mRNAs and on rRNA under certain conditions and that also participates in 3Ј maturation of 16S rRNA (1,5,6). Other studies have found that RNase R also can complement CsdA, a DEAD box helicase, at low temperatures (7). RNase R is a large (99 kDa), multidomain protein that contains two RNA binding cold shock domains in its N-terminal region, another RNA binding S1 domain in its C-terminal region, and a central nuclease domain. RNase R is subject to a variety of regulatory processes, and its cellular localization and stability are affected by its N-and C-terminal regions (7-11). One of the more interesting properties of RNase R is the presence of an intrinsic RNA helicase activity (11,12). RNase R and polynucleotide phosphorylase are the primary nucleases involved in degradation of structured RNAs in vivo (1, 13). Polynucleotide phosphorylase degrades structured RNA as a part of the RNA degradosome that is associated with an RNA helicase (14 -16). In contrast, RNase R appears to act by itself, although the mechanism by which it degrades structured RNA and the role of its intrinsic helicase activity in this process are not yet fully understood.In earlier work (11), we examined the RNase R helicase activity and found that it is dependent on ATP binding, but not hydrolysis, and that ATP binding occurs only in the presence of a double-stranded RNA substrate. We identified ATP-binding Walker A and Walker B motifs in Escherichia coli RNase R and found that they were conserved in 88% of mesophilic bacterial genera analyzed but were absent from thermophilic bacteria. We also found that although the nuclease acti...

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