Faus I, Richardson J P
Department of Chemistry, Indiana University, Bloomington 47405.
Biochemistry. 1989 Apr 18;28(8):3510-7. doi: 10.1021/bi00434a054.
Termination of transcription at tR1, the rho-dependent terminator between genes cro and cII of bacteriophage lambda, is mediated by interactions between rho protein and an RNA sequence element called rut. We show, using a filter retention assay technique, that rho protein binds with about 10-fold lower affinity to variants of cro RNA lacking both parts of rut or to normal cro RNA having one or the other part of rut bound to a complementary DNA oligonucleotide than it binds to unmodified cro RNA. These same variant and modified forms are nearly devoid of the strong rho ATPase cofactor activity of cro RNA. Estimates of binding energies of the rho-cro RNA interaction under different conditions reveal that termination function correlates with about 12.6 kcal of binding energy, of which two-thirds is due to nonelectrostatic interactions. The rut segment is shown to contribute about 1 kcal, nearly all to nonelectrostatic interactions. KCl is found to be more effective than potassium glutamate as a competitive counterion, and a decrease in 1.4 kcal of binding energy due to counterion competition correlates with a loss of termination and ATPase activities. In sum, the results indicate that the rut sequence contributes substantially to the overall binding affinity, that ionic interactions are also important, and that mere binding of rho to RNA is not sufficient for rho ATPase activation.
噬菌体λ的cro基因和cII基因之间的ρ因子依赖性终止子tR1处的转录终止，是由ρ蛋白与一个称为rut的RNA序列元件之间的相互作用介导的。我们使用滤膜滞留测定技术表明，ρ蛋白与缺乏rut两个部分的cro RNA变体或与rut的一个或另一个部分与互补DNA寡核苷酸结合的正常cro RNA的结合亲和力，比与未修饰的cro RNA的结合亲和力低约10倍。这些相同的变体和修饰形式几乎没有cro RNA强大的ρATP酶辅因子活性。在不同条件下对ρ-cro RNA相互作用的结合能估计表明，终止功能与约12.6千卡的结合能相关，其中三分之二归因于非静电相互作用。rut片段显示贡献约1千卡，几乎全部归因于非静电相互作用。发现KCl作为竞争性抗衡离子比谷氨酸钾更有效，并且由于抗衡离子竞争导致的1.4千卡结合能的降低与终止和ATP酶活性的丧失相关。总之，结果表明rut序列对整体结合亲和力有很大贡献，离子相互作用也很重要，并且仅ρ蛋白与RNA的结合不足以激活ρATP酶。
