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首页> 外文期刊>Journal of bacteriology >Roles of catabolite activator protein sites centered at -81.5 and -41.5 in the activation of the Klebsiella aerogenes histidine utilization operon hutUH.
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Roles of catabolite activator protein sites centered at -81.5 and -41.5 in the activation of the Klebsiella aerogenes histidine utilization operon hutUH.

机译：分解代谢因子激活蛋白的位点集中在-81.5和-41.5，在产气克雷伯菌组氨酸利用操纵子hutUH的激活中。

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摘要

The Klebsiella aerogenes hutUH operon is preceded by a promoter region, hut(P), that contains two divergent promoters (hutUp and Pc) which overlap and are alternately expressed. In the absence of the catabolite gene activator protein-cyclic AMP (CAP-cAMP) complex, Pc is predominantly expressed while hutUp is largely repressed. CAP-cAMP has the dual effect of repressing transcription from Pc while simultaneously activating transcription from hutUp. DNA deletion mutations in this region were used to identify DNA sequences required for transcription of these two promoters. We showed that inactivation of Pc by DNA deletion did not result in activation of hutUp in vitro or in vivo. In addition, Escherichia coli CAP mutants that are known to bind and bend DNA normally but are unable to activate various CAP-dependent promoters were also unable to activate hutUp in vivo. These results invalidate an indirect activation model by which CAP-mediated repression of Pc in itself would led to activation of hutUp. Gel retardation asays with various deletion mutations of hut(P) and DNase I protection analyses revealed a high-affinity CAP binding site (CAP site 1) centered at -81.5 relative to the hutUp start of transcription and a second low-affinity CAP site (CAP site 2) centered at about -41.5. CAP site 1 is essential for activation of hutUp. Although CAP site 2 by itself is unable to activate hutUp in vivo under catabolite-activating conditions, it appears to be required for maximal transcription from a site centered at -41.5, does not activate hutUp suggests that the role of CAP-cAMP at the weaker CAP site may be different from that of other promoters containing a similarly positioned site. We propose that CAP directly stimulates the activity of RNA polymerase at hutUp and that this reaction is completely dependent on a naturally occurring CAP site centered at -81.5 and also involves a second CAP site centered at about -41.5 for maximal activation.
机译：产气克雷伯氏菌hutUH操纵子之前是一个启动子区域hut（P），其中包含两个重叠且交替表达的两个不同的启动子（hutUp和Pc）。在没有分解代谢物基因激活剂蛋白-环AMP（CAP-cAMP）复合物的情况下，Pc主要表达，而hutUp却被大量抑制。 CAP-cAMP具有抑制Pc转录同时激活hutUp转录的双重作用。该区域中的DNA缺失突变用于鉴定这两个启动子转录所需的DNA序列。我们表明，通过DNA缺失使PC失活不会在体外或体内激活hutUp。另外，已知能正常结合和弯曲DNA但是不能激活各种CAP依赖性启动子的大肠杆菌CAP突变体也不能在体内激活hutUp。这些结果使间接激活模型无效，间接激活模型通过CAP介导的Pc抑制本身将导致hutUp激活。胶凝阻滞作用与hut（P）的各种缺失突变和DNase I保护分析揭示了相对于hutUp转录起点和第二个低亲和力CAP位点的高亲和力CAP结合位点（CAP位点1）位于-81.5。 CAP网站2）的中心约为-41.5。 CAP站点1对于激活hutUp是必不可少的。尽管CAP位点2本身无法在分解代谢物激活条件下体内激活hutUp，但似乎需要从以-41.5为中心的位点进行最大转录，而不能激活hutUp则表明CAP-cAMP在较弱的位置CAP位点可能不同于其他包含相似位置的启动子。我们建议，CAP直接刺激hutUp处的RNA聚合酶的活性，并且该反应完全取决于以-81.5为中心的天然CAP位点，并且还涉及以大约-41.5为中心的第二个CAP位点以实现最大活化。

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