title: Binding of nucleic acids to E. coli RNase HI observed by NMR and CD spectroscopy.
abstract: To clarify the mechanism by which the RNA portion of a DNA/RNA hybrid is specifically hydrolyzed by ribonuclease H (RNase H), the binding of a DNA/RNA hybrid, a DNA/DNA duplex, or an RNA/RNA duplex to RNase HI from Escherichia coli was investigated by 1H-15N heteronuclear NMR. Chemical shift changes of backbone amide resonances were monitored while the substrate, a hybrid 9-mer duplex, a DNA/DNA 12-mer duplex, or an RNA/RNA 12-mer duplex was titrated. The amino acid residues affected by the addition of each 12-mer duplex were almost identical to those affected by the substrate hybrid binding, and resided close to the active site of the enzyme. The results reveal that all the duplexes, hybrid-, DNA-, and RNA-duplex, bind to the enzyme. From the linewidth analysis of the resonance peaks, it was found that the exchange rates for the binding were different between the hybrid and the other duplexes. The NMR and CD data suggest that conformational changes occur in the enzyme and the hybrid duplex upon binding.
authors.affiliation: Protein Engineering Research Institute, Osaka, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Oda
authors.forename: S
authors.initials: S
authors.lastname: Iwai
authors.forename: E
authors.initials: E
authors.lastname: Ohtsuka
authors.forename: M
authors.initials: M
authors.lastname: Ishikawa
authors.forename: M
authors.initials: M
authors.lastname: Ikehara
authors.forename: H
authors.initials: H
authors.lastname: Nakamura
mesh_terms: D001483:Base Sequence; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004563:Electrochemistry; D004926:Escherichia coli / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D016914:Ribonuclease H / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D012329:RNA, Bacterial; D016914:Ribonuclease H; C078856:ribonuclease HI