Mülsch A, Colpan M, Wollny E, Gassen H G, Riesner D
Nucleic Acids Res. 1981 May 25;9(10):2367-85. doi: 10.1093/nar/9.10.2367.
The interaction of the ribosomal protein S1 from E. coli MRE 600 with oligonucleotides was studied by hydrodynamic, spectrophotometric, and kinetic methods. UV-difference spectra which are induced by the complex formation could be separated into a hyperchromic contribution originating from the nucleic acid moiety and a hypochromic contribution from the protein. Systematic determination of binding and rate constants was carried out by the temperature-jump relaxation technique. From the quantitative evaluation of the relaxation times and the relaxation amplitudes, the following conclusions could be drawn: The stoichiometry of the complex formation is one mole S1 per one mole oligonucleotide. The binding constant K, the recombination rate constant kR, and the dissociation rate constant kD, respectively, were measured at different temperatures. The values at 10 degrees C are K = 2 x 10(6) M-1, kR = 1.3 x 10(8) M-1S-1, kD = 65 s-1 for A(pA) 12 and K = 7.5 x 10(5) M-1, kR = 6.8 x 10(7) M-1S-1, kD = 90 S-1 for U(pU) 12. Discrepancies with data reported elsewhere are discussed. The stacking-unstacking equilibrium of the free oligonucleotide is frozen if the oligonucleotide is bound to the protein. The conformational change of the oligonucleotide does not occur in the form of a preequilibrium, but is induced after the primary binding step.
采用流体力学、分光光度法和动力学方法研究了来自大肠杆菌MRE 600的核糖体蛋白S1与寡核苷酸的相互作用。由复合物形成诱导的紫外差光谱可分为源自核酸部分的增色贡献和来自蛋白质的减色贡献。通过温度跳跃弛豫技术系统地测定了结合常数和速率常数。通过对弛豫时间和弛豫幅度的定量评估,可以得出以下结论:复合物形成的化学计量比是每摩尔寡核苷酸一摩尔S1。分别在不同温度下测量了结合常数K、重组速率常数kR和解离速率常数kD。对于A(pA)12,10℃时的值为K = 2×10(6) M-1,kR = 1.3×10(8) M-1S-1,kD = 65 s-1;对于U(pU)12,K = 7.5×10(5) M-1,kR = 6.8×10(7) M-1S-1,kD = 90 S-1。讨论了与其他地方报道的数据的差异。如果寡核苷酸与蛋白质结合,游离寡核苷酸的堆积-解堆积平衡就会被冻结。寡核苷酸的构象变化不是以预平衡的形式发生,而是在初级结合步骤之后被诱导。