A NEW EXPERIMENTAL APPROACH FOR STUDYING THE ASSOCIATION BETWEEN RNA-POLYMERASE AND THE TET PROMOTER OF PBR322
A NEW EXPERIMENTAL APPROACH FOR STUDYING THE ASSOCIATION BETWEEN RNA-POLYMERASE AND THE TET PROMOTER OF PBR322
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DOI：
10.1093/nar/12.3.1697
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发表时间：
1984-01-01
影响因子：
14.9
通讯作者：
DAUNE, M
中科院分区：
文献类型：
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作者：
BERTRANDBURGGRAF, E;LEFEVRE, JF;DAUNE, M
为了跟踪大肠杆菌RNA聚合酶启动转录的动力学，我们使用了Mc Clure(1980)(7)所描述的流产启动过程。代替放射性标记，我们利用荧光探针(UTP伽马ANS)快速准确地测定转录速率，并从动力学方程中推导出经典两步模型的结合常数(Kb)和异构化速率(K2)。对线性化的pBR322启动子进行了分析，并研究了温度(从25℃到37℃)和pH(从6到8.3)的作用。这种缔合是由熵驱动的(增量H度=29千卡/摩尔，增量S度=130E)。异构化反应的活化能为13千卡/摩尔。K2和k-2均随pH值增大而增大。KBK2产物对pH不敏感的原因可以用聚合酶与其特定部位结合的过程性来解释。
In order to follow the kinetics of the initiation of transcription by the E. coli RNA polymerase, we have used the procedure of abortive initiation as described by Mc Clure (1980) (7). In place of radioactive labeling we have taken advantage of a fluorescent probe (UTP gamma ANS) to obtain fast and accurate determinations of the rate of transcription and to deduce from kinetic equations both the binding constant (KB) and the rate of isomerization (k2) which characterize the classical two-step model. This analysis was applied to the tet promoter of pBR322 in a linearized plasmid DNA and was studied in function of temperature (from 25 degrees C to 37 degrees C) and of pH (from 6 to 8.3). The association is entropy driven (delta H degrees = 29 Kcal/mole and delta S degrees = 130 e.u.). The activation energy of isomerization is 13 Kcal/mole. Both k2 and k-2 are increasing with pH. The insensitivity to pH of the KBK2 product could be tentatively explained in terms of the processive aspect of the polymerase binding to its specific site.