ANALYSES OF THE DNA-BINDING AND TRANSCRIPTIONAL ACTIVATION PROPERTIES OF EXSA, THE TRANSCRIPTIONAL ACTIVATOR OF THE PSEUDOMONAS-AERUGINOSA EXOENZYME-S REGULON
ANALYSES OF THE DNA-BINDING AND TRANSCRIPTIONAL ACTIVATION PROPERTIES OF EXSA, THE TRANSCRIPTIONAL ACTIVATOR OF THE PSEUDOMONAS-AERUGINOSA EXOENZYME-S REGULON
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DOI：
10.1128/jb.177.15.4427-4436.1995
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发表时间：
1995-08-01
影响因子：
3.2
通讯作者：
FRANK, DW
中科院分区：
文献类型：
--
作者：
HOVEY, AK;FRANK, DW
ExsA被认为是铜绿假单胞菌胞外酶S产生的中心调节因子。在这项研究中，ExsA的DNA结合和转录激活特性进行了研究。ExsA作为融合蛋白MALA 3A 2产生并纯化，其显示特异性结合调节外切酶S反式调节基因座（pC）和直接位于exsA下游的基因座（pD）的转录的启动子区。以前，MALA 3A 2显示结合exoS 5' PstI-NsiI区域，其含有两个独立但协调调节的（ExsA介导的）启动子，pS'（现在称为pORF 1）和pS。DNA酶I的足迹分析的启动子区域结合ExsA揭示了一个共同的保护TXAAAAXA的共识序列。共有序列位于pD、pS和pORF 1转录起始位点上游-51至-52 bp处。启动子融合，DNA结合，和诱变分析表明，共识序列是重要的转录激活。每个ExsA控制的启动子区含有至少两个紧密接近的共有位点，类似于AraC控制的（大肠杆菌）或VirF控制的（小肠结肠炎耶尔森氏菌）启动子中所见的半位点的排列。然而，这项研究的结果表明，只有一个共识位点是必需的外切酶S（pS）或ORF 1启动子（pORF 1）启动转录。这些数据表明，外切酶S调节子的成员可以被定义为具有ExsA共有元件，其定位在相对于转录起始位点的bp -51或-52处。
ExsA has been implicated as a central regulator of exoenzyme S production by Pseudomonas aeruginosa. In this study, the DNA-binding and transcriptional activation properties of ExsA were investigated. ExsA was produced and purified as a fusion protein, MALA3A2, which was shown to bind specifically to promoter regions that regulated transcription of the exoenzyme S trans-regulatory locus (pC) and a locus located directly downstream of exsA (pD). Previously, MALA3A2 was shown to bind the exoS 5' PstI-NsiI region, which contained two independent but coordinately regulated (ExsA-mediated) promoters, pS' (now termed pORF1) and pS. DNase I footprint analysis of the promoter regions bound by ExsA revealed a common protected consensus sequence of TXAAAAXA. The consensus sequence was located -51 to -52 bp upstream of the transcriptional start sites for pD, pS, and pORF1. Promoter fusion, DNA-binding, and mutagenesis analyses indicated that the consensus sequence was important for transcriptional activation. Each ExsA-controlled promoter region contained at least two consensus sites in close proximity, similar to the arrangement of half-sites seen in AraC controlled (Escherichia coli) or VirF-controlled (Yersinia enterocolitica) promoters. However, the results of this study suggested that only one consensus site was required in the exoenzyme S (pS) or ORF1 promoter (pORF1) to initiate transcription. These data suggest that members of the exoenzyme S regulon can be defined as possessing an ExsA consensus element which maps at bp -51 or -52 relative to the transcriptional start site.