Johnson R S, Chester R E
Department of Biochemistry, East Carolina University School of Medicine, Greenville, NC, 27858, USA.
J Mol Biol. 1998 Oct 23;283(2):353-70. doi: 10.1006/jmbi.1998.2101.
We have conducted a detailed kinetic and thermodynamic analysis of open complex formation between Escherichia coli RNA polymerase and the A1 promoter from bacteriophage T7 by monitoring alterations in the intrinsic protein fluorescence of RNA polymerase in stopped-flow kinetic studies. The stopped-flow kinetic data are consistent with a minimal model involving four steps for the formation of the open complex. Arrhenius plots for both the association and dissociation reactions for the equilibrium binding step leading to the formation of the closed complex were linear. With a positive van't Hoff enthalpy (DeltaHobs=18(+/-3) kcal mol-1) and a positive entropy (DeltaSobs=94(+/-15) e.u.) change for the equilibrium binding process, formation of the closed complex is entropy driven. The value of the apparent association rate constant for this binding step was approximately three orders of magnitude less than that expected for facilitated binding. Thus, a minimum of two steps is required to describe the formation of the closed complex. A fast facilitated binding step appears to be followed by a conformational change in RNA polymerase which leads to the formation of the closed complex. A non-linear Arrhenius plot obtained for the isomerization step in the conversion of the closed complex to an open one indicates that there are at least two steps in the conversion of the closed complex to an open one. We have assigned the apparent activation energy of 9.1(+/-1.9) kcal mol-1 to the step involving a conformational change in the protein and nucleation of strand separation and the apparent activation energy of 46(+/-12) kcal mol-1 to the step involving strand separation. At 37 degreesC, the value of the macroscopic isomerization rate constant (0.26(+/-0.02) s-1) in the conversion of the closed complex to an open one was an order of magnitude greater than the value reported in abortive initiation assays. This suggests that open complex formation is not the rate-determining step in the initiation of transcription in the case of the A1 promoter. To gain greater insight into the mechanism of initiation at the A1 promoter, we investigated the process of abortive product formation (pppApU) under conditions of non-saturating concentrations of the initiating nucleotide. A comparison of the lag times in the approach to the steady-state rate of abortive product formation when the reaction was initiated by the addition of UTP, ATP, the enzyme and the A1 promoter, respectively, indicates that the initiating nucleotide plays a key regulatory role in the initiation of transcription in the case of the A1 promoter.
我们通过在停流动力学研究中监测RNA聚合酶内在蛋白荧光的变化，对大肠杆菌RNA聚合酶与噬菌体T7的A1启动子之间开放复合物的形成进行了详细的动力学和热力学分析。停流动力学数据与一个涉及开放复合物形成的四个步骤的最小模型一致。导致封闭复合物形成的平衡结合步骤的缔合和解离反应的阿累尼乌斯图均为线性。对于平衡结合过程，其范特霍夫焓变正值（ΔHobs = 18（±3）kcal mol-1）和熵变正值（ΔSobs = 94（±15）e.u.），封闭复合物的形成是由熵驱动的。该结合步骤的表观缔合速率常数的值比促进结合预期的值小约三个数量级。因此，描述封闭复合物的形成至少需要两个步骤。一个快速的促进结合步骤之后似乎是RNA聚合酶的构象变化，这导致了封闭复合物的形成。从封闭复合物转变为开放复合物的异构化步骤得到的非线性阿累尼乌斯图表明，封闭复合物转变为开放复合物至少有两个步骤。我们将9.1（±1.9）kcal mol-1的表观活化能赋予涉及蛋白质构象变化和链分离成核的步骤，将46（±12）kcal mol-1的表观活化能赋予涉及链分离的步骤。在37℃时，封闭复合物转变为开放复合物的宏观异构化速率常数的值（0.26（±0.02）s-1）比流产起始测定中报道的值大一个数量级。这表明在A1启动子的情况下，开放复合物的形成不是转录起始的速率决定步骤。为了更深入了解A1启动子处的起始机制，我们研究了在起始核苷酸非饱和浓度条件下流产产物形成（pppApU）的过程。当分别通过添加UTP、ATP、酶和A1启动子引发反应时，对达到流产产物形成稳态速率的滞后时间进行比较，表明在A1启动子的情况下，起始核苷酸在转录起始中起关键调节作用。
