Sullivan J J, Bjornson K P, Sowers L C, deHaseth P L
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4935, USA.
Biochemistry. 1997 Jul 1;36(26):8005-12. doi: 10.1021/bi970363k.
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.
人们已投入大量精力研究大肠杆菌RNA聚合酶与启动子DNA在形成开放复合物过程中的结合动力学。链分离发生在约12个碱基对的区域，包括转录起始位点。然而，由于缺乏一种能够检测开放复合物形成的灵敏实时方法，这些努力受到了极大阻碍。在此，我们使用短的（86 bp）合成启动子，其在变为单链的区域带有2-氨基嘌呤（2-AP）取代，以光谱法监测开放复合物的形成。我们证明，带有取代的启动子在链分离区域和起始位点选择方面的行为与仅含四种常见碱基的启动子相似。发现开放复合物的形成会产生增强的荧光信号，其发射最大值具有2-氨基嘌呤的特征。这种用于检测开放复合物形成的光谱测定法被发现非常适合研究强启动子，使用停流装置可在数秒的时间尺度上跟踪开放复合物的形成。在 -35区域引入两个额外的非保守碱基对，得到了一个开放复合物形成速度慢100倍的启动子。相同的底物也用于监测RNA合成起始后发生的启动子重新退火。在本研究中使用的两种启动子变体中观察到该过程的速率相似。
