In vitro transcription of the histidine utilization (hutUH) operon from Klebsiella aerogenes.
In vitro transcription of the histidine utilization (hutUH) operon from Klebsiella aerogenes.
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产气克雷伯菌组氨酸利用 (hutUH) 操纵子的体外转录。
DOI：
10.1128/jb.173.1.116-123.1991
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发表时间：
1991
影响因子：
3.2
通讯作者：
Bender,RA
中科院分区：
文献类型：
--
作者：
Osuna,R;Boylan,SA;Bender,RA
产气克雷伯氏菌中hutUH操纵子之前的启动子区域含有两个方向相反的重叠启动子。在不存在分解代谢物基因激活蛋白-环AMP（CAP-cAMP）的情况下，转录主要从其功能尚未确定的后向启动子（Pc）进行，并且仅非常弱地从前向hutUH启动子hutUp进行。在CAP-cAMP的存在下，Pc被抑制，并且从hutUp的转录是有利的。从K.产气菌的蛋白质，并显示与大肠杆菌的相应蛋白质在功能上是可互换的，这表明大肠杆菌。coliRNAP可用于研究hut转录的某些方面。我们发现，hutUp的逐渐激活（通过增加CAP，cAMP或甘油的浓度）导致了Pc的平行抑制，支持两个启动子之间的直接竞争。与CAP结合位点相似的DNA序列的存在和集中在核苷酸-82（相对于hutUp）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，在Pc处形成开放复合物的相对缓慢表明Pc是弱启动子，并且可能是RNAP的弱竞争者。观察到的Pc超过hutUp的优势表明后者是一个甚至更弱的启动子。因此，Pc的抑制不足以引起观察到的hutUp活性的增加，并且CAP-cAMP复合物必须在hutUp的激活中起直接作用。
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.