T7 RNA polymerase-induced bending of promoter DNA is coupled to DNA opening
T7 RNA polymerase-induced bending of promoter DNA is coupled to DNA opening
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DOI：
10.1021/bi0522910
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发表时间：
2006-04-18
期刊：
影响因子：
2.9
通讯作者：
Patel, SS
中科院分区：
文献类型：
--
作者：
Tang, GQ;Patel, SS
为了启动转录，T7RNA聚合酶(RNAP)与其启动子DNA形成一个特定的复合体，并在起始点附近熔化几个碱基对形成开放的复合体。以前的凝胶电泳研究表明，起始复合体中的启动子DNA是弯曲的[Ujvari，A.，和Martin，C.T.(2000)J.Mol。比奥尔。295,1173-1184]。在这里，我们使用荧光共振能量转移(FRET)来研究T7RNAP的闭合和开放复合体中启动子DNA的构象。我们已经使用稳态和时间分辨的荧光方法来测量双染料标记的双链启动子和预熔的气泡启动子中的FRET效率。当双链启动子与T7 RNAP形成复合体时，FRET效率的变化很小，因此DNA端到端距离的变化很小。另一方面，当气泡启动子与T7 RNAP结合时，或者当启动核苷酸被添加到双链启动子-T7 RNAP复合体中时，FRET的变化相对较大。DNA端到端距离的缩短表明DNA在泡状DNA复合体和双链启动子与起始核苷酸的复合体中发生弯曲。我们的结果与这样的模型是一致的，即在没有起始核苷酸的情况下，存在闭合和开放的复合体的分布，并且启动子DNA轻微弯曲
To initiate transcription, T7 RNA polymerase (RNAP) forms a specific complex with its promoter DNA and melts several base pairs near the initiation site to form an open complex. Previous gel electrophoresis studies have indicated that the promoter DNA in the initiation complex is bent [Ujvari, A., and Martin, C. T. (2000) J. Mol. Biol. 295, 1173-1184]. Here we use fluorescence resonance energy transfer (FRET) to investigate the conformation of promoter DNA in the closed and open complexes of T7 RNAP. We have used steady state and time-resolved fluorescence approaches to measure the FRET efficiency in a doubly dye-labeled duplex promoter and in a premelted bubble promoter. Changes in the FRET efficiency and hence the DNA end-to-end distance changes are small when the duplex promoter forms a complex with T7 RNAP. On the other hand, FRET changes are relatively larger when the bubble promoter binds T7 RNAP or when initiating nucleotides are added to the duplex promoter-T7 RNAP complex. The shortening of DNA end-to-end distances is indicative of DNA bending in the bubble DNA complex and in the duplex promoter complex with the initiating nucleotides. Our results are consistent with the model in which in the absence of initiating nucleotides there is a distribution of closed and open complexes, and the promoter DNA is bent slightly by