Binding of Escherichia coli RNA polymerase holoenzyme to bacteriophage T7 DNA. Measurements of the rate of open complex formation at T7 promoter A.
Binding of Escherichia coli RNA polymerase holoenzyme to bacteriophage T7 DNA. Measurements of the rate of open complex formation at T7 promoter A.
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大肠杆菌 RNA 聚合酶全酶与噬菌体 T7 DNA 的结合。
DOI：
10.1016/0022-2836(82)90490-9
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发表时间：
1982
影响因子：
5.6
通讯作者：
Chamberlin,MJ
中科院分区：
文献类型：
--
作者：
Rosenberg,S;Kadesch,TR;Chamberlin,MJ
我们采用了一种基于Chamberlin等人(1979)定量转录分析的新方法，研究了大肠杆菌RNA聚合酶全酶与噬菌体T7DNA上A1启动子在体外形成开放启动子复合体的速率。该程序测量了在一个反应中实现恒定的RNA合成速度所需的时间，在该反应中，每个RNA聚合酶分子被限制为单轮转录。这一时间与活性RNA聚合酶分子形成开放复合体的速度成反比，前提是RNA链的启动足够快。在有利于在T7启动子A1处形成开放启动子复合体的溶液条件下，观察到的速率与过滤结合、利福平挑战或模板竞争等其他方法获得的速率一致。然而，该程序也适用于许多其他方法不能使用的情况。这包括低温或离子强度升高等反应条件，在这些条件下，开放的启动子复合体不稳定。使用这一方法的研究，以及基于模板竞争实验的测量(Kadesch等人，1982，附文)产生了T7A1启动子上RNA聚合酶结合和激活的主要步骤的大致情况。在30℃以上，模板缔合和开放络合物的形成速度都很快(t1 2~10~15 S)，对温度或一价阳离子或二价阳离子浓度的适度变化不敏感。开放复合体形成的速率常数不会因酶或DNA浓度的显著变化而改变。因此，在30℃时的速率不受启动子熔化的限制，而可能是由早期的瞬时启动子复合体的形成所限制的。模板结合速率和开放复合体形成速率的密切比较表明，在闭合启动子复合体和开放启动子复合体之间可能存在额外的中间体。在低于25℃的温度下，开放复合体的形成速度大大降低，而模板缔合的速率保持相对不变；因此，积累的复合体具有闭合启动子复合体的性质。在15℃时，开放络合物的形成速度对反离子浓度也很敏感。因此，对于T7A1启动子，似乎在低温下限速步骤不同，涉及闭合启动子复合体的熔融/异构化为开放启动子复合体。因此，即使对于单个启动子位点，也可以根据转录条件的不同，在从游离RNA聚合酶到延长RNA聚合酶的途径中进行不止一步的速率决定。
We have studied the rate of open promoter complex formation in vitro between Escherichia coli RNA polymerase holoenzyme and the A 1 promoter on bacteriophage T7 DNA, employing a novel method based on the quantitative transcription assay of Chamberlin et al.(1979). The procedure measures the time needed to achieve a constant rate of RNA synthesis in a reaction in which each RNA polymerase molecule is restricted to a single round of transcription. This time is inversely proportional to the rate of open complex formation by active RNA polymerase molecules, provided that RNA chain initiation is sufficiently rapid. Under solution conditions that favor formation of open promoter complexes at T7 promoter A 1 at equilibrium, the rates observed are in good agreement with those obtained using other methods such as filter binding, rifampicin challenge or template competition. However, the procedure is also applicable in many situations where other methods cannot be used. This includes reaction conditions such as low temperatures or elevated ionic strengths, where open promoter complexes are not stable. Studies using this procedure, taken with measurements based on template competition experiments (Kadesch et al., 1982, accompanying paper) yield a general picture of the major steps in the binding and activation of RNA polymerase at the T7 A 1 promoter. Above 30° C, the rates of template association and open complex formation are both rapid (t 1 2~ 10 to 15 s), and are insensitive to moderate changes in temperature or the concentration of monovalent or divalent cations. The rate constant for open complex formation is not altered by substantial changes in enzyme or DNA concentration. Thus the rate at 30° C is not limited by promoter melting, but possibly by formation of an early, transient promoter complex. Close comparison of the rates of template association and of open complex formation suggests that there may be an additional intermediate between the closed promoter complex and the open promoter complex. At temperatures below 25° C, the rate of open complex formation decreases substantially, while the rate of template association remains relatively unchanged; hence, a complex accumulates having the properties of a closed promoter complex. The rate of open complex formation at 15° C also becomes sensitive to the counterion concentration. Thus for the T7 A 1 promoter, it appears that the rate-limiting step is different at low temperatures, and involves the melting/isomerization of the closed promoter complex to the open promoter complex. Thus, even for a single promoter site, more than one step can be rate determining in the pathway from free RNA polymerase to elongating RNA polymerase, depending on the conditions employed for transcription.