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首页> 外文期刊>Journal of bacteriology >In vitro transcription of the histidine utilization (hutUH) operon from Klebsiella aerogenes.
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In vitro transcription of the histidine utilization (hutUH) operon from Klebsiella aerogenes.

机译：产气克雷伯氏菌的组氨酸利用（hutUH）操纵子的体外转录。

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The promoter region preceding the hutUH operon in Klebsiella aerogenes contains two oppositely oriented, overlapping promoters. In the absence of catabolite gene activator protein-cyclic AMP (CAP-cAMP), transcription proceeds primarily from the backward-oriented promoter (Pc), whose function has not yet been determined, and only very weakly from the forward hutUH promoter, hutUp. In the presence of CAP-cAMP, Pc is repressed and transcription from hutUp is favored. Two protein components required for this in vitro transcription system, RNA polymerase (RNAP) and CAP, were purified from K. aerogenes and were shown to be functionally interchangeable with the corresponding proteins from Escherichia coli, suggesting that E. coli RNAP could be used to study some aspects of hut transcription. We showed that a gradual activation of hutUp (by increasing concentrations of CAP, cAMP, or glycerol) resulted in a parallel repression of Pc, arguing in favor of a direct competition between the two promoters. The presence of a DNA sequence resembling the consensus for CAP-binding sites and centered at nucleotide -82 (relative to hutUp) initially suggested that a primary role of CAP was to repress Pc, thereby indirectly activating hutUp. However, the relatively slow formation of open complexes at Pc, even in the absence of CAP-cAMP, showed that Pc is a weak promoter and likely to be a poor competitor for RNAP. The observed dominance of Pc over hutUp suggested that the latter is an even weaker promoter. Thus, repression of Pc would not be sufficient to cause the observed increase in hutUp activity, and the CAP-cAMP complex must play a direct role in the activation of hutUp.
机译：产气克雷伯氏菌中hutUH操纵子之前的启动子区域包含两个方向相反的重叠启动子。在没有分解代谢物基因激活蛋白环状AMP（CAP-cAMP）的情况下，转录主要从尚未确定其功能的向后启动子（Pc）进行，而仅从正向hutUH启动子hutUp进行。在CAP-cAMP的存在下，Pc被阻遏并且有利于从hutUp转录。从产气假单胞菌中纯化了该体外转录系统所需的两种蛋白质成分，即RNA聚合酶（RNAP）和CAP，并证明它们与大肠杆菌中的相应蛋白质具有功能上的互换性，这表明大肠杆菌RNAP可用于研究小屋转录的某些方面。我们显示hutUp的逐渐激活（通过增加CAP，cAMP或甘油的浓度）导致Pc的平行阻遏，主张两个启动子之间的直接竞争。 DNA序列的存在类似于CAP结合位点的共有序列，并以核苷酸-82（相对于hutUp）为中心，最初表明CAP的主要作用是抑制PC，从而间接激活hutUp。然而，即使在没有CAP-cAMP的情况下，Pc上开放复合物的形成也相对缓慢，这表明Pc是弱启动子，可能是RNAP的较弱竞争者。观察到的Pc在hutUp上的优势表明，后者是一个更弱的启动子。因此，抑制Pc不足以导致观察到的hutUp活性增加，并且CAP-cAMP复合物必须在hutUp的激活中起直接作用。

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