Yamanaka K, Ishihama A, Nagata K
Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan.
J Biol Chem. 1990 Jul 5;265(19):11151-5.
Reconstitution of influenza virus nucleoprotein (NP)-RNA complexes was performed with segment 8 RNA, which was synthesized in vitro from cDNA, and NP purified from virions. Under optimum conditions established using a filter binding assay and a gel retardation assay, NP was found to bind any RNA longer than 15 nucleotides. NP-RNA complexes formed at 30 degrees C are more resistant to high concentrations of NaCl than those formed at 0 degrees C. Treatment of NP with N-ethylmaleimide gave no effect on its RNA binding activity, whereas treatment with alkaline phosphatase enhanced its RNA binding activity. The newly developed "reverse-printing" method of RNase V1-treated complexes revealed that reconstituted NP-RNA complexes carry RNase V1-sensitive sites as do native ribonucleoprotein (RNP) cores (RNA polymerase-NP-RNA complexes), implying that RNA-NP complexes structurally similar to native RNP cores are reconstituted from isolated components.
利用从 cDNA 体外合成的第 8 节段 RNA 和从病毒粒子中纯化的核蛋白（NP）进行流感病毒核蛋白（NP）-RNA 复合物的重组。在使用滤膜结合测定法和凝胶阻滞测定法确定的最佳条件下，发现 NP 能结合任何长度超过 15 个核苷酸的 RNA。30℃形成的 NP-RNA 复合物比 0℃形成的复合物对高浓度 NaCl 更具抗性。用 N-乙基马来酰亚胺处理 NP 对其 RNA 结合活性没有影响，而用碱性磷酸酶处理则增强了其 RNA 结合活性。新开发的 RNase V1 处理复合物的“反向印迹”方法表明，重组的 NP-RNA 复合物与天然核糖核蛋白（RNP）核心（RNA 聚合酶-NP-RNA 复合物）一样带有 RNase V1 敏感位点，这意味着从分离的组分中重组出了结构上与天然 RNP 核心相似的 RNA-NP 复合物。
