Spectroscopic determination of open complex formation at promoters for Escherichia coli RNA polymerase.
Spectroscopic determination of open complex formation at promoters for Escherichia coli RNA polymerase.
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DOI：
10.1021/bi970363k
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发表时间：
1997-07
期刊：
影响因子：
2.9
通讯作者：
J. Sullivan;K. Bjornson;L. Sowers;P. Dehaseth
中科院分区：
文献类型：
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作者：
J. Sullivan;K. Bjornson;L. Sowers;P. Dehaseth
人们在研究大肠杆菌RNA聚合酶与启动子DNA结合形成开放复合体的动力学方面做了大量的工作。链分离发生在大约12个碱基对上，包括转录起始点。然而，由于缺乏一种灵敏的、实时的方法来检测开放复合体的形成，这些努力受到了极大的阻碍。在这里，我们使用短的(86bp)合成启动子，在成为单链的区域带有2-氨基嘌呤(2-AP)取代，以光谱监测开放复合体的形成。我们证明了带有取代的启动子在链分离区域和起始点选择方面的行为类似于那些只含有四个共同碱基的启动子。开放络合物的形成产生了增强的荧光信号，其最大发射特征为2-氨基嘌呤。这种开放复合体形成的光谱分析被发现非常适合于强促进剂的研究，允许利用停流装置在几秒钟的时间尺度上跟踪开放复合体的形成。在-35区域引入另外两个非共识碱基对，导致启动子开放复合体的形成速度减慢100倍。同样的底物也被用来监测启动RNA合成后的启动子重新退火。在这项研究中使用的两个启动子变异体的这一过程中观察到了相似的比率。
A considerable amount of effort has been expended studying the kinetics of association of Escherichia coli RNA polymerase with promoter DNA in forming the open complex. Strand separation occurs over about 12 base pairs and includes the transcription start site. However, these efforts have been significantly hampered by the lack of a sensitive, real time method by which formation of an open complex could be assayed. Here, we employ short (86 bp) synthetic promoters with 2-aminopurine (2-AP) substitutions in the region that becomes single-stranded to spectroscopically monitor open complex formation. We demonstrate that promoters bearing the substitutions behave in a manner similar to that of those containing only the four common bases with respect to both the region of strand separation and start site selection. Open complex formation was found to yield an increased fluorescence signal with an emission maximum characteristic of 2-aminopurine. This spectroscopic assay for open complex formation was found to be well-suited to the investigation of a strong promoter, allowing open complex formation to be followed over a time scale of seconds with a stopped flow apparatus. The introduction of two additional nonconsensus base pairs in the -35 region resulted in a promoter for which open complex formation was 100-fold slower. The same substrates were also used to monitor the promoter re-annealing that ensues upon initiation of RNA synthesis. Similar rates for this process were observed for the two promoter variants employed in this study.