Kumar A, Patel S S
Department of Biochemistry, Ohio State University, Columbus 43210, USA.
Biochemistry. 1997 Nov 11;36(45):13954-62. doi: 10.1021/bi971432y.
The mechanism of transcription repression of T7 RNA polymerase by T7 lysozyme was investigated using a combination of kinetic and equilibrium methods. HPLC gel-filtration experiments demonstrated complex formation between T7 lysozyme, T7 RNA polymerase, and promoter DNA. The interactions between the two proteins were quantitated by measuring in real time the changes in protein fluorescence upon binary complex formation using stopped-flow kinetics. Complex formation between T7 lysozyme and the RNA polymerase was found to occur by a one-step process, with a bimolecular association rate constant of 38 microM-1 S-1 and a dissociation rate constant of 3.5 S-1. These constants provided an equilibrium dissociation constant, Kd, of 92 nM for the polymerase lysozyme complex. The interactions of the polymerase with the DNA were studied by stopped-flow kinetics and nitrocellulose equilibrium DNA binding experiments in the absence and in the presence of T7 lysozyme. The results showed that T7 lysozyme did not prevent or change the kinetic or thermodynamic interactions of the RNA polymerase with the DNA. T7 lysozyme by itself did not bind to the DNA, but since it bound to the RNA polymerase as well as to the polymerase DNA complex, transcription repression must involve the formation of the ternary complex between T7 lysozyme, T7 RNA polymerase and the promoter DNA. The effect of T7 lysozyme was most striking on runoff product synthesis which was greatly inhibited whereas the steady-state synthesis of abortive products, limited by polymerase cycling or RNA dissociation, was relatively unaffected by the presence of T7 lysozyme. Investigation of the pre-steady-state kinetics of transcription in the presence and absence of T7 lysozyme indicated that the inhibition of runoff product synthesis was largely due to inhibition of transcription initiation and transition from initiation to elongation.
运用动力学和平衡方法相结合的方式，对T7溶菌酶抑制T7 RNA聚合酶转录的机制进行了研究。高效液相色谱凝胶过滤实验证明了T7溶菌酶、T7 RNA聚合酶和启动子DNA之间形成了复合物。通过使用停流动力学实时测量二元复合物形成时蛋白质荧光的变化，对这两种蛋白质之间的相互作用进行了定量分析。发现T7溶菌酶与RNA聚合酶之间的复合物形成是一个一步过程，双分子缔合速率常数为38 μM⁻¹ s⁻¹，解离速率常数为3.5 s⁻¹。这些常数得出聚合酶 - 溶菌酶复合物的平衡解离常数Kd为92 nM。在不存在和存在T7溶菌酶的情况下，通过停流动力学和硝酸纤维素平衡DNA结合实验研究了聚合酶与DNA的相互作用。结果表明，T7溶菌酶不会阻止或改变RNA聚合酶与DNA的动力学或热力学相互作用。T7溶菌酶本身不与DNA结合，但由于它与RNA聚合酶以及聚合酶 - DNA复合物结合，转录抑制必定涉及T7溶菌酶、T7 RNA聚合酶和启动子DNA之间三元复合物的形成。T7溶菌酶对径流产物合成的影响最为显著，径流产物合成受到极大抑制，而流产产物的稳态合成受聚合酶循环或RNA解离限制，相对不受T7溶菌酶存在的影响。对存在和不存在T7溶菌酶时转录的稳态前动力学研究表明，径流产物合成的抑制主要是由于转录起始以及从起始到延伸的转变受到抑制。
