Osuna R, Janes B K, Bender R A
Department of Biology, University of Michigan, Ann Arbor 48109-1048.
J Bacteriol. 1994 Sep;176(17):5513-24. doi: 10.1128/jb.176.17.5513-5524.1994.
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.
产气克雷伯菌的hutUH操纵子之前有一个启动子区域，即hut(P)，它包含两个方向相反的启动子（hutUp和Pc），这两个启动子相互重叠且交替表达。在缺乏分解代谢基因激活蛋白 - 环磷酸腺苷（CAP - cAMP）复合物的情况下，Pc主要表达，而hutUp大部分被抑制。CAP - cAMP具有双重作用，既能抑制Pc的转录，又能同时激活hutUp的转录。利用该区域的DNA缺失突变来鉴定这两个启动子转录所需的DNA序列。我们发现，通过DNA缺失使Pc失活在体外或体内均未导致hutUp的激活。此外，已知能正常结合并弯曲DNA但无法激活各种CAP依赖性启动子的大肠杆菌CAP突变体在体内也无法激活hutUp。这些结果否定了一种间接激活模型，即CAP介导的对Pc的抑制本身会导致hutUp的激活。对hut(P)的各种缺失突变进行凝胶阻滞分析和DNase I保护分析表明，存在一个高亲和力的CAP结合位点（CAP位点1），其相对于hutUp转录起始点位于 - 81.5处，以及第二个低亲和力的CAP位点（CAP位点2），其中心位于约 - 41.5处。CAP位点1对于hutUp的激活至关重要。尽管在分解代谢激活条件下，CAP位点2本身在体内无法激活hutUp，但它似乎是从位于 - 41.5处的位点进行最大转录所必需的，不激活hutUp表明CAP - cAMP在较弱的CAP位点的作用可能与其他含有类似位置位点的启动子不同。我们提出，CAP直接刺激hutUp处RNA聚合酶的活性，并且该反应完全依赖于位于 - 81.5处的天然存在的CAP位点，并且还涉及位于约 - 41.5处的第二个CAP位点以实现最大激活。
