Synthesis and Use of Modified Oligonucleotides for the Study on a Substrate Recognition Site of RNase H

修饰寡核苷酸的合成及其在 RNase H 底物识别位点研究中的应用

• 批准号: 01571134
• 负责人: INOUE Hideo
• 金额: $ 1.22万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01571134/
• 关键词: E. coli RNase H; RNA-DNA hybrid; Photoaffinity labeling; Photoactive oligodeoxynucleotide; Crosslinking; Lysyl endopeptidase; Substrate recognition site; 大腸菌RNase H; RNA・DNAハイブリド; 大腸菌RNaseH

Photoaffinity labeling of a substrate recognition site of E. Coli RNase H has been studied with photolabile RNA-DNA hybrid substrates. A phenyldiazirine derivative was introduced to DNA oligomer (5'-GTCATCTCC-3') at the 5'-end or the inside. In the latter case, the diazirine group was attached to C2'-modified uridine residue having an amino-linker arm which was introduced instead of T.Irradiation of a mixture of the photoactive hybrid (the DNA was labeled with ^<32>P) and RNase H gave crosslinked product(s). The product could be separated from RNase H by cation-exchange chromatography, although it was contaminated with starting nucleic acids. The affinity-labeled product from each reaction was digested with lysyl endopeptidase. The resultant peptides were separated by reverse-phase HPLC. Peptide-mapping analysis of labeled RNase Hs showed that crosslinking reactions had occurred at peptide LEP 2, 3 and 4. It has been reported that Asp 10, Glu48 and Asp 70 were crucial for E. coli RNase H activity (these amino acid residues are located at the above peptide fragments). These data suggest that LEP 2, 3 and 4 constitute the substrate recognition site of the enzyme.

光亲和标记E.已经用光不稳定的RNA-DNA杂交底物研究了Coli RNase H。在DNA寡聚体（5 ′-GTCATCTCC-3 ′）的5 ′-末端或内部引入苯基二氮杂环丙烷衍生物。在后一种情况下，将二氮杂环丙烯基团连接至具有氨基-接头臂的C2 '-修饰的尿苷残基，所述氨基-接头臂被引入而不是T。辐照光敏杂交体（DNA用13 P标记<32>）和RNA酶H的混合物得到交联产物。产物可以通过阳离子交换层析从RNase H分离，尽管它被起始核酸污染。用赖氨酰内肽酶消化来自每个反应的亲和标记产物。通过反相高效液相色谱法分离所得肽。标记的RNase Hs的肽图谱分析表明，交联反应发生在肽LEP 2，3和4。据报道，Asp 10、Glu 48和Asp 70是大肠杆菌的关键酶。coli RNase H活性（这些氨基酸残基位于上述肽片段上）。这些数据表明LEP 2、3和4构成酶的底物识别位点。