The non-RNase H domain of Saccharomyces cerevisiae RNase H1 binds double-stranded RNA: magnesium modulates the switch between double-stranded RNA binding and RNase H activity.
Eukaryotic ribonucleases H of known sequence are composed of an RNase H domain similar in size and sequence to that of Escherichia coli RNase HI and additional domains of unknown function. The RNase H1 of Saccharomyces cerevisiae has such an RNase H domain at its C-terminus. Here we show that the N-terminal non-RNase H portion of the yeast RNase H1 binds tightly to double-stranded RNA (dsRNA) and RNA-DNA hybrids even in the absence of the RNase H domain. Two copies of a sequence with limited similarity to the dsRNA-binding motif are present in this N-terminus. When the first of these sequences is altered, the protein no longer binds tightly to dsRNA and exhibits an increase in RNase H activity. Unlike other dsRNA-binding proteins, increasing the Mg2+ concentration from 0.5 mM to 5 mM inhibits binding of RNase H1 to dsRNA; yet a protein missing the RNase H domain binds strongly to dsRNA even at the higher Mg2+ concentration. These results suggest that binding to dsRNA and RNase H activity are mutually exclusive, and the Mg2+ concentration is critical for switching between the activities. Changes in the Mg2+ concentration or proteolytic severing of the dsRNA-binding domain could alter the activity or location of the RNase H and may govern access of the enzyme to the substrate. Sequences similar to the dsRNA-binding motif are present in other eukaryotic RNases H and the transactivating protein of cauliflower mosaic virus, suggesting that these proteins may also bind to dsRNA.
D000595:Amino Acid Sequence; D001665:Binding Sites; D015971:Gene Expression Regulation, Enzymologic; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D011070:Poly I-C / Q000378:metabolism; D011485:Protein Binding; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012331:RNA, Fungal / Q000378:metabolism*; D016914:Ribonuclease H / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D009692:Nucleic Acid Heteroduplexes; D012330:RNA, Double-Stranded; D012331:RNA, Fungal; D016914:Ribonuclease H; C078856:ribonuclease HI; D008274:Magnesium; D011070:Poly I-C