Characterization in vitro of the effect of spacer length on the activity of Escherichia coli RNA polymerase at the TAC promoter.
Characterization in vitro of the effect of spacer length on the activity of Escherichia coli RNA polymerase at the TAC promoter.
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发表时间：
1985-03
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通讯作者：
M. E. Mulligan;J. Brosius;W. R. McClure
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作者：
M. E. Mulligan;J. Brosius;W. R. McClure
我们描述了三种杂合trp-lacUV5 (TAC)启动子的体外表征，它们与-10和-35大肠杆菌启动子共有六聚体序列具有完美的同源性，但在-10和-35区域之间的距离不同。三个启动子 TAC16、TAC17 和 TAC18 的间距分别为 16、17 和 18 个碱基对。我们测量了这三个启动子上的 KB 和 k2（描述开放复合物形成的常数）。我们还测量了它们对超螺旋质粒 DNA 的相对强度。我们的结果表明，这些是迄今为止体外表征的最强启动子，并证实了共有启动子序列是“最佳”的假设。我们发现 TAC17 启动子 (KBk2 = 8.4 X 10(7) M-1 s-1) 比 TAC16 (KBk2 大约 1.5 X 10(7) M-1 s-1) 或 TAC18 (KBk2 大约 3.5 X 10(7) M-1 s-1) 启动子更强，这一结果与关于间隔区长度影响的其他发现一致。转录起始点的选择受间隔区长度的影响。所有启动子的转录均在中等浓度的盐（低于 150 mM）下受到刺激，并在高盐浓度（300 mM）下持续存在。
We describe the characterization in vitro of three hybrid trp-lacUV5 (TAC) promoters, which have perfect homology to the -10 and -35 Escherichia coli promoter consensus hexamer sequences, but which differ in the distance between the -10 and -35 regions. The three promoters, TAC16, TAC17, and TAC18 have spacings of 16, 17, and 18 base pairs, respectively. We have measured KB and k2, the constants that describe the formation of open complexes, on these three promoters. We have also measured their relative strengths on supercoiled plasmid DNA. Our results show that these are the strongest promoters that have been characterized in vitro so far and confirm the hypothesis that the consensus promoter sequence is "best." We find the TAC17 promoter (KBk2 = 8.4 X 10(7) M-1 s-1) to be stronger than either the TAC16 (KBk2 approximately 1.5 X 10(7) M-1 s-1) or TAC18 (KBk2 approximately 3.5 X 10(7) M-1 s-1) promoters, a result that is in agreement with other findings on the effect of spacer length. The choice of start point for transcription is affected by spacer length. Transcription from all the promoters was stimulated at moderate concentrations of salt (less than 150 mM) and persisted at high salt concentrations (300 mM).