Abstract
RNase R, which belongs to the RNB family of enzymes, is a 3' to 5' hydrolytic exoribonuclease able to digest highly structured RNA. It was previously reported that RNase R possesses an intrinsic helicase activity that is independent of its ribonuclease activity. However, the properties of this helicase activity and its relationship to the ribonuclease activity were not clear. Here, we show that helicase activity is dependent on ATP and have identified ATP-binding Walker A and Walker B motifs that are present in Escherichia coli RNase R and in 88% of mesophilic bacterial genera analyzed, but absent from thermophilic bacteria. We also show by mutational analysis that both of these motifs are required for helicase activity. Interestingly, the Walker A motif is located in the C-terminal region of RNase R, whereas the Walker B motif is in its N-terminal region implying that the two parts of the protein must come together to generate a functional ATP-binding site. Direct measurement of ATP binding confirmed that ATP binds only when double-stranded RNA is present. Detailed analysis of the helicase activity revealed that ATP hydrolysis is not required because both adenosine 5'-O-(thiotriphosphate) and adenosine 5'-(β,γ-imino)triphosphate can stimulate helicase activity, as can other nucleoside triphosphates. Although the nuclease activity of RNase R is not needed for its helicase activity, the helicase activity is important for effective nuclease activity against a dsRNA substrate, particularly at lower temperatures and with more stable duplexes. Moreover, competition experiments and mutational analysis revealed that the helicase activity utilizes the same catalytic channel as the nuclease activity. These findings indicate that the helicase activity plays an essential role in the catalytic efficiency of RNase R.
Highlights
Escherichia coli RNase R contains an intrinsic RNA helicase activity
We show that helicase activity is dependent on ATP and have identified ATP-binding Walker A and Walker B motifs that are present in Escherichia coli RNase R and in 88% of mesophilic bacterial genera analyzed, but absent from thermophilic bacteria
RNase R contains a central nuclease domain and four putative RNA-binding domains: two cold-shock domains (CSD1 and CSD2) near the N terminus of the protein, an S1 domain near the C terminus of the protein, and a low complexity, highly basic region at the C terminus (Fig. 1A) [3]. Earlier work from another group indicated that RNase R has weak RNA helicase activity that depends on cold shock domain 2 and that the helicase activity is unaffected when the nuclease activity is inactivated, suggesting that the two activities are independent [15]
Summary
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. Our laboratory suggested that it was due to natural thermal breathing and tight binding of the RNA substrate at the bottom of the catalytic channel within the nuclease domain We hypothesized that such binding forced the substrate to translocate by one nucleotide every catalytic cycle and concomitantly opened one base pair as the enzyme digested through the structured region of RNA [3, 4]. Competition experiments and mutational analysis strongly suggest that both the helicase and nuclease activities are carried out in the same catalytic channel These findings indicate that the helicase activity plays an important role in the catalytic properties of RNase R
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