Romaniuk P J
Nucleic Acids Res. 1985 Jul 25;13(14):5369-87. doi: 10.1093/nar/13.14.5369.
A nitrocellulose filter binding assay has been developed to study the interaction of Xenopus transcription factor IIIA with 5S RNA. The protein binds Xenopus oocyte 5S RNA with an association constant of 1.4 X 10(9) M-1 at 0.1 M salt, pH 7.5 at 20 degrees C. TF IIIA binds wheat germ 5S RNA with a two-fold higher affinity, E. coli 5S RNA with a four-fold weaker affinity, and has a barely detectable interaction with yeast tRNAphe. The preference for binding eukaryotic 5S RNA is enhanced in competition assays. The homologous reconstituted complex contains one molecule each of protein and 5S RNA and is indistinguishable from native 7S RNP in mobility on non-denaturing polyacrylamide gels. The conformation of the RNA in reconstituted particles is identical to the conformation of RNA in native 7S RNP. Further analysis of the homologous interaction reveals that complex formation is a favoured both by enthalpy and entropy. The 5S RNA binding activity has a broad pH optimum spanning pH 6.0 to pH 8.0. Determination of the salt dependence of Ka reveals that as many as 5 lysine-phosphate type ionic bonds may be formed in the homologous complex. Approximately 68% of the free energy of complex formation is contributed by non-electrostatic interactions between TF IIIA and Xenopus 5S RNA.
已开发出一种硝酸纤维素滤膜结合试验，用于研究非洲爪蟾转录因子IIIA与5S RNA的相互作用。在20℃、0.1M盐、pH 7.5条件下，该蛋白与非洲爪蟾卵母细胞5S RNA结合的缔合常数为1.4×10⁹M⁻¹。TF IIIA与小麦胚芽5S RNA的结合亲和力高两倍，与大肠杆菌5S RNA的结合亲和力弱四倍，与酵母苯丙氨酸tRNA的相互作用几乎检测不到。在竞争试验中，对结合真核生物5S RNA的偏好性增强。同源重组复合物含有一个蛋白分子和一个5S RNA分子，在非变性聚丙烯酰胺凝胶上的迁移率与天然7S RNP无法区分。重组颗粒中RNA的构象与天然7S RNP中RNA的构象相同。对同源相互作用的进一步分析表明，复合物形成在焓和熵方面都受到青睐。5S RNA结合活性在pH 6.0至pH 8.0范围内有较宽的最佳pH值。对Ka的盐依赖性测定表明，在同源复合物中可能形成多达5个赖氨酸 - 磷酸型离子键。复合物形成的自由能中约68%由TF IIIA与非洲爪蟾5S RNA之间的非静电相互作用贡献。
