Interaction of sigma factor sigmaN with Escherichia coli RNA polymerase core enzyme.
Interaction of sigma factor sigmaN with Escherichia coli RNA polymerase core enzyme.
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西格玛因子 sigmaN 与大肠杆菌 RNA 聚合酶核心酶的相互作用。
DOI：
10.1042/bj3520539
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
J. Hoggett
中科院分区：
文献类型：
--
作者：
D. Scott;A. L. Ferguson;M. Gallegos;M. Pitt;M. Buck;J. Hoggett
使用生物合成标记的 7-氮杂色氨酸- (7AW)sigma(N) 研究了 DNA 依赖性 RNA 聚合酶 (RNAP) sigma(N)-全酶的平衡结合和组装动力学。即使存在高浓度的其他含色氨酸蛋白质，此类 7AW 蛋白质的光谱特性也可以选择性地监测其吸光度和荧光。 7AWsigma(N) 在刺激 sigma(N) 特异性启动子转录以及与大肠杆菌核心 RNAP 结合的体外凝胶电泳测定中保留了其生物活性。此外，sigma(N) 的五个 Trp-->Ala 单突变体显示出支持氮限制条件下的生长，并且在体内激活 sigma(N) 依赖性 nifH 启动子方面表现出相当的效率，表明没有一个色氨酸残基对于活性而言是必需的。通过分析超速离心检查 7AWsigma(N) 与核心 RNAP 的平衡结合。在沉降平衡实验中，315 nm 处的吸光度数据（选择性报告游离和结合 7AWsigma(N) 的分布）确定形成了 1:1 复合物，解离常数低于 2 µM。使用停流荧光光度法研究了 7AWsigma(N) 和核心 RNAP 之间相互作用的动力学。当样品在 280 nm 处激发时，观察到荧光强度的双相降低，而在 315 nm 处仅观察到两相中较慢的一个。根据 sigma(N) 与核心 RNAP 的快速双分子缔合之后是相对缓慢的异构化步骤的机制来分析动力学数据。讨论了这些发现对大肠杆菌中 sigma(N) 和主要 sigma 因子 sigma(70) 之间竞争的影响。
The equilibrium binding and kinetics of assembly of the DNA-dependent RNA polymerase (RNAP) sigma(N)-holoenzyme has been investigated using biosynthetically labelled 7-azatryptophyl- (7AW)sigma(N). The spectroscopic properties of such 7AW proteins allows their absorbance and fluorescence to be monitored selectively, even in the presence of high concentrations of other tryptophan-containing proteins. The 7AWsigma(N) retained its biological activity in stimulating transcription from sigma(N)-specific promoters, and in in vitro gel electrophoresis assays of binding to core RNAP from Escherichia coli. Furthermore, five Trp-->Ala single mutants of sigma(N) were shown to support growth under conditions of nitrogen limitation, and showed comparable efficiency in activating the sigma(N)-dependent nifH promoter in vivo, indicating that none of the tryptophan residues were essential for activity. The equilibrium binding of 7AWsigma(N) to core RNAP was examined by analytical ultracentrifugation. In sedimentation equilibrium experiments, absorbance data at 315 nm (which reports selectively on the distribution of free and bound 7AWsigma(N)) established that a 1:1 complex was formed, with a dissociation constant lower than 2 microM. The kinetics of the interaction between 7AWsigma(N) and core RNAP was investigated using stopped-flow spectrofluorimetry. A biphasic decrease in fluorescence intensity was observed when samples were excited at 280 nm, whereas only the slower of the two phases was observed at 315 nm. The kinetic data were analysed in terms of a mechanism in which a fast bimolecular association of sigma(N) with core RNAP is followed by a relatively slow isomerization step. The consequences of these findings on the competition between sigma(N) and the major sigma factor, sigma(70), in Escherichia coli are discussed.
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用色氨酸类似物增强蛋白质光谱：蛋白质-蛋白质和蛋白质-核酸相互作用的荧光光谱。
影响因子：
--
作者：
Ross,JB;Szabo,AG;Hogue,CW
通讯作者：
Hogue,CW
影响因子：
3.4
作者：
JOHNSON, ML;CORREIA, JJ;HALVORSON, HR
通讯作者：
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DOI：
10.1073/pnas.82.24.8453
发表时间：
1985-12-01
影响因子：
11.1
作者：
HUNT, TP;MAGASANIK, B
通讯作者：
MAGASANIK, B
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影响因子：
2.9
作者：
Rusinova,E;Ross,JB;Laue,TM;Sowers,LC;Senear,DF
通讯作者：
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