The Helicase Activity of Ribonuclease R Is Essential for Efficient Nuclease Activity.
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.
From the Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, Florida 33101.
Sk Tofajjen
ST
Hossain
From the Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, Florida 33101.
Arun
A
Malhotra
From the Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, Florida 33101. Electronic address: m.deutscher@med.miami.edu.
Murray P
MP
Deutscher
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D020816:Amino Acid Motifs; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005095:Exoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012330:RNA, Double-Stranded; C489683:rnr protein, E coli; D000255:Adenosine Triphosphate; D005095:Exoribonucleases; D020365:RNA Helicases
ATP; Escherichia coli (E. coli); RNA degradation; RNA helicase; ribonuclease