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首页> 外文期刊>Biophysical Chemistry: An International Journal Devoted to the Physical Chemistry of Biological Phenomena >CONFORMATIONAL CHANGES OF E-COLI RNA POLYMERASE DURING TRANSCRIPTION INITIATION
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CONFORMATIONAL CHANGES OF E-COLI RNA POLYMERASE DURING TRANSCRIPTION INITIATION

机译：转录起始过程中大肠杆菌核糖核酸聚合酶的构象变化

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Escherichia coli RNA polymerase-promoter complex undergoes a multistep process to initiate transcription. We have employed fluorescence spectroscopic approaches to detect the conformational states of the enzyme during this multistep process. A fluorescence assay based on the measurement of fluorescence of free and promoter-bound enzyme as a function of temperature within the range of 4 to 37 degrees C showed that, starting with initial 'closed complex', there are conformationally two distinct intermediate states of the polymerase till it attains the final form required for transcription initiation. The equilibrium from closed complex (RP(c)) to open complex (RP(o)) consists of at least the following two intermediate complexes: [GRAPHICS] Higher order structure of RNAP in each of these complexes was probed by means of measurement of accessibilities of the tryptophan fluorophores to the acrylamide. In the next part of the study, TbGTP, a fluorescent substrate, has been used to probe the state of active site in the enzyme for the complexes RP(c), RP(il), RP(i2) and RP(o), respectively. From the comparison of changes in the parameters such as, fluorescence polarization anisotropy of TbGTP and its accessibility to the neutral quencher, acrylamide, in free and promoter-bound enzyme, we have further substantiated the first part of our results. Together these results suggest that formations of RP(c) and RP(il) do not involve radical conformational changes in the enzyme, while the enzyme undergoes major change in conformation in the steps RP(il) --> RP(i2) and RP(i2) --> RP(o). The strong tryptophan promoter cloned in plasmid pDR720 was chosen as a model promoter in these studies. [References: 39]
机译：大肠杆菌RNA聚合酶启动子复合物经历了多步过程以启动转录。我们已采用荧光光谱方法来检测此多步过程中酶的构象状态。基于游离和启动子结合的酶的荧光随温度在4至37摄氏度范围内的函数进行测量的荧光分析表明，从初始的“封闭复合物”开始，构象上存在两个不同的中间状态聚合酶直至达到转录起始所需的最终形式。从封闭复合物（RP（c））到开放复合物（RP（o））的平衡至少由以下两个中间复合物组成：[GRAPHICS]通过测量H2O3的含量，探索了每种复合物中RNAP的高级结构。色氨酸荧光团对丙烯酰胺的可及性。在研究的下一部分中，荧光底物TbGTP已用于探测复合物RP（c），RP（il），RP（i2）和RP（o）的酶中活性位点的状态，分别。通过比较TbGTP的荧光偏振各向异性及其在游离酶和启动子结合酶中对中性猝灭剂丙烯酰胺的可及性等参数变化的比较，我们进一步证实了结果的第一部分。这些结果共同表明，RP（c）和RP（il）的形成不涉及酶的自由基构象变化，而在步骤RP（il）-> RP（i2）和RP中，酶的构象发生重大变化（i2）-> RP（o）。在这些研究中，选择克隆到质粒pDR720中的强色氨酸启动子作为模型启动子。 [参考：39]

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