通过核磁共振（NMR）和圆二色光谱（CD）观察核酸与大肠杆菌核糖核酸酶HI的结合。
Binding of nucleic acids to E. coli RNase HI observed by NMR and CD spectroscopy.
作者信息
Oda Y, Iwai S, Ohtsuka E, Ishikawa M, Ikehara M, Nakamura H
机构信息
Protein Engineering Research Institute, Osaka, Japan.
出版信息
Nucleic Acids Res. 1993 Oct 11;21(20):4690-5. doi: 10.1093/nar/21.20.4690.
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.
为阐明核糖核酸酶H（RNase H）特异性水解DNA/RNA杂交体中RNA部分的机制，通过1H-15N异核核磁共振研究了DNA/RNA杂交体、DNA/DNA双链体或RNA/RNA双链体与大肠杆菌RNase HI的结合。在滴定底物（杂交9聚体双链体、DNA/DNA 12聚体双链体或RNA/RNA 12聚体双链体）时，监测主链酰胺共振的化学位移变化。添加每种12聚体双链体所影响的氨基酸残基与受底物杂交体结合影响的氨基酸残基几乎相同，且位于酶的活性位点附近。结果表明，所有双链体，即杂交双链体、DNA双链体和RNA双链体，均与该酶结合。通过共振峰的线宽分析发现，杂交体与其他双链体之间的结合交换速率不同。核磁共振和圆二色光谱数据表明，结合时酶和杂交双链体会发生构象变化。
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