The occupation of the final sigma(54)-dependent Pu promoter of Pseudomonas putida by the integration host factor (IHF) under different growth conditions has been monitored in its native state and stoichiometry (i.e. monocopy) with UV laser footprinting technology. We present evidence that an abrupt change in intracellular IHF concentrations occurs when P. putida cells enter stationary phase. This change results in enhanced binding of the factor to the promoter and in the ensuing bending of the target DNA. Since Pu activity depends rigorously on DNA bending, promoter occupation is in turn translated into a much higher transcriptional output when cells leave exponential growth. Inspection of the residual activity of Pu in an IHF(-) strain reveals that IHF predominantly locks the capacity of the promoter to specific growth stages and also that additional physiological signals are entered in the system through final sigma(54)-RNA polymerase. The results substantiate the notion that final sigma(54) promoters process metabolic co-regulation signals through factor-induced changes in the architecture of the cognate DNA region. Further, they validate UV laser technology as a suitable tool to visualize nondisruptive alterations of DNA shape in vivo.

译文

在不同的生长条件下,整合宿主因子 (IHF) 对恶臭假单胞菌的最终sigma(54) 依赖性Pu启动子的占领已通过UV激光足迹技术以其天然状态和化学计量 (即单倍) 监测。我们提供的证据表明,当P. putida细胞进入固定相时,细胞内IHF浓度会发生突然变化。这种变化导致该因子与启动子的结合增强,并导致靶DNA随之弯曲。由于Pu活性严格依赖于DNA弯曲,因此当细胞离开指数生长时,启动子的占领又转化为更高的转录输出。对IHF(-) 菌株中Pu的残留活性的检查表明,IHF主要将启动子的能力锁定在特定的生长阶段,并且还通过最终的sigma(54)-RNA聚合酶进入系统中。结果证实了最终的sigma(54) 启动子通过因子诱导的同源DNA区域结构变化来处理代谢协同调节信号的概念。此外,他们还验证了UV激光技术是可视化体内DNA形状非破坏性改变的合适工具。

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