Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-lambda PR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening.
Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-lambda PR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening.
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大肠杆菌 RNA 聚合酶-lambda PR 启动子相互作用的速率常数的温度依赖性。
DOI：
10.1016/0022-2836(85)90293-1
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发表时间：
1985
影响因子：
5.6
通讯作者：
RecordJr,MT
中科院分区：
文献类型：
--
作者：
Roe,JH;Burgess,RR;RecordJr,MT
用硝酸纤维素滤膜结合法研究了大肠杆菌RNA聚合酶（R）与λ PR启动子（P）之间开放复合物（RPO）的形成和解离动力学。的动力学数据提供了进一步的证据的机制R+ P的I1的I2的I2的RP O，其中I1和I2是动力学上可区分的中间体配合物，在这个促进剂，不积累的反应条件下调查。总的二级缔合速率常数（ka）随着温度的升高而显著增加，产生在20 kcal（接近37° C）至40 kcal（接近13° C）范围内的温度依赖性活化能（1 kcal= 4.184 kJ）。两个异构化步骤（I1 → I2和I2 → RP 0）似乎高度依赖于温度。除了在低温（< 13° C）下，步骤I1 → I2（我们将其归因于聚合酶中具有大的负δ Cpo值的构象变化）在所研究的反应物浓度下是限速的，因此对假一级缔合反应的表观活化能做出主要贡献。随后的步骤I2 → RP O，我们将其归因于DNA解链，具有较高的活化能（超过100 kcal），但仅在低温（< 13° C）下才变得限速。在研究的所有条件下，初始结合步骤R+ P托地I 1似乎在异构化反应的时间尺度上处于平衡状态;该步骤的平衡常数不是温度的强函数，在测定的标准离子条件（40 m m-Tris HCl（pH 8.0），10 m m-MgCl 2，0.12 m-KCl）下约为10 7 m− 1。在低温下，解离反应的活化能变得越来越负，范围从37° C附近的约-9大卡到13° C附近的-30大卡。因此，形成开放复合物的热力学（van 't霍夫）能谱Δ Ho很大，且与温度有关，当温度从37 ° C降低到13° C时，Δ Ho从大约29 kcal增加到70 kcal。相应的ΔC p o值约为− 2.4 kcal/deg。我们认为，这个大的负Δ Cpo值主要是由于在RNA聚合酶的关键第二步构象变化（I1 → I2）中疏水表面的掩埋所致。开放复合物形成的三步机制表明启动子分为两类：顺序R+ P→ k1 I1 → k2 + I2 → k3 → RP 0和快速结合平衡R+ P顺序K1 I1 → K2 I2 → K3 RP 0。这两类的伪一阶时间常数τ obs（τ obs=（k a R T）− 1+ k 2− 1+ k 3− 1）仅在对ka的解释上不同，因此在低聚合酶浓度下的行为上不同（ka = k 1（顺序）; ka = K 1 k 2（快速平衡））。在高聚合酶浓度下，两个类别的τ obs值由两个异构化步骤中的较慢步骤决定，其通常对温度和其他调节变量的响应不同。探讨了这种机械分解在体内和体外的各种启动子的行为的后果。
The kinetics of formation and of dissociation of open complexes (RP O) between Escherichia coli RNA polymerase (R) and the λP R promoter (P) have been studied as a function of temperature in the physiological range using the nitrocellulose filter binding assay. The kinetic data provide further evidence for the mechanism R+ P⇋ I 1⇋ I 2⇋ RP O, where I 1 and I 2 are kinetically distinguishable intermediate complexes at this promoter which do not accumulate under the reaction conditions investigated. The overall second-order association rate constant (k a) increases dramatically with increasing temperature, yielding a temperature-dependent activation energy in the range 20 kcal (near 37° C) to 40 kcal (near 13° C)(1 kcal= 4.184 kJ). Both isomerization steps (I 1→ I 2 and I 2→ RP 0) appear to be highly temperature dependent. Except at low temperatures (< 13° C) the step I 1→ I 2, which we attribute to a conformational change in the polymerase with a large negative δC p o value, is rate-limiting at the reactant concentrations investigated and hence makes the dominant contribution to the apparent activation energy of the pseudo first-order association reaction. The subsequent step I 2→ RP O, which we attribute to DNA melting, has a higher activation energy (in excess of 100 kcal) but only becomes rate-limiting at low temperature (< 13° C). The initial binding step R+ P⇋ I 1 appears to be in equilibrium on the time-scale of the isomerization reactions under all conditions investigated; the equilibrium constant for this step is not a strong function of temperature and is approximately 10 7 m− 1 under the standard ionic conditions of the assay (40 m m-Tris HCl (pH 8.0), 10 m m-MgCl 2, 0.12 m-KCl). The activation energy of the dissociation reaction becomes increasingly negative at low temperatures, ranging from approximately− 9 kcal near 37° C to− 30 kcal near 13° C. Thermodynamic (van't Hoff) enthalpies ΔH o of open complex formation consequently are large and temperature-dependent, increasing from approximately 29 to 70 kcal as the temperature is reduced from 37 to 13° C. The corresponding ΔC p o value is approximately− 2.4 kcal/deg. We propose that this large negative ΔC p o value arises primarily from the burial of hydrophobic surface in the conformational change (I 1⇋ I 2) in RNA polymerase in the key second step of the mechanism. The three-step mechanism for open complex formation suggests a division of promoters into two classes: sequential R+ P→ k 1 I 1→ k 2+ I 2→ k 3→ RP 0 and rapid binding equilibrium R+ P⇋ K 1 I 1→ K 2 I 2→ K 3 RP 0. The pseudo first-order time constants τ obs for these two classes (τ obs=(k a R T)− 1+ k 2− 1+ k 3− 1) differ only in the interpretation of k a, and hence in their behavior at low polymerase concentration (k a= k 1 (sequential); k a= K 1 k 2 (rapid equilibrium)). At high polymerase concentration, values of τ obs for both classes are determined by the slower of the two isomerization steps, which in general respond differently to temperature and other regulatory variables. Consequences of this mechanistic decomposition for the behavior of various promoters in vivo and in vitro are explored.