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嗜盐古菌中组成型启动子和调控型启动子的体内分析。

In vivo analyses of constitutive and regulated promoters in halophilic archaea.

作者信息

Gregor Dagmar, Pfeifer Felicitas

机构信息

Institut für Mikrobiologie und Genetik, Technische Universität Darmstadt, Schnittspahnstr. 10, D-64287 Darmstadt, Germany.

出版信息

Microbiology (Reading). 2005 Jan;151(Pt 1):25-33. doi: 10.1099/mic.0.27541-0.

Abstract

The two gvpA promoters P(cA) and P(pA) of Halobacterium salinarum, and the P(mcA) promoter of Haloferax mediterranei were investigated with respect to growth-phase-dependent expression and regulation in Haloferax volcanii transformants using the bgaH reading frame encoding BgaH, an enzyme with beta-galactosidase activity, as reporter. For comparison, the P(fdx) promoter of the ferredoxin gene of Hbt. salinarum and the P(bgaH) promoter of Haloferax lucentense (formerly Haloferax alicantei) were analysed. P(fdx), driving the expression of a house-keeping gene, was highly active during the exponential growth phase, whereas P(bgaH) and the three gvpA promoters yielded the largest activities during the stationary growth phase. Compared to P(fdx), the basal promoter activities of P(pA) and P(mcA) were rather low, and larger activities were only detected in the presence of the endogenous transcriptional activator protein GvpE. The P(cA) promoter does not yield a detectable basal promoter activity and is only active in the presence of the homologous cGvpE. To investigate whether the P(cA)-TATA box and the BRE element were the reason for the lack of the basal P(cA) activity, these elements and also sequences further upstream were substituted with the respective sequences of the stronger P(pA) promoter and investigated in Hfx. volcanii transformants. All these promoter chimera did not yield a detectable basal promoter activity. However, whenever the P(pA)-BRE element was substituted for the P(cA)-BRE, an enhanced cGvpE-mediated activation was observed. The promoter chimeras harbouring P(pA)-BRE plus 5 (or more) bp further upstream also gained activation by the heterologous pGvpE and mcGvpE proteins. The sequence required for the GvpE-mediated activation was determined by a 4 bp scanning mutagenesis with the 45 bp region upstream of P(mcA)-BRE. None of these alterations influenced the basal promoter activity, but the sequence TGAAACGG-n4-TGAACCAA was important for the GvpE-mediated activation of P(mcA).

摘要

利用编码具有β-半乳糖苷酶活性的BgaH的bgaH读码框作为报告基因,研究了盐生盐杆菌的两个gvpA启动子P(cA)和P(pA)以及地中海嗜盐嗜热栖热菌的P(mcA)启动子在沃氏嗜盐栖热菌转化体中的生长阶段依赖性表达和调控。作为比较,分析了盐生盐杆菌铁氧化还原蛋白基因的P(fdx)启动子和透明嗜盐栖热菌(以前称为阿利坎特嗜盐栖热菌)的P(bgaH)启动子。驱动管家基因表达的P(fdx)在指数生长期高度活跃,而P(bgaH)和三个gvpA启动子在稳定生长期产生最大活性。与P(fdx)相比,P(pA)和P(mcA)的基础启动子活性相当低,只有在内源转录激活蛋白GvpE存在时才能检测到更大的活性。P(cA)启动子不产生可检测的基础启动子活性,仅在同源cGvpE存在时才活跃。为了研究P(cA)-TATA框和BRE元件是否是P(cA)缺乏基础活性的原因,用更强的P(pA)启动子的相应序列替换了这些元件以及更上游的序列,并在沃氏嗜盐栖热菌转化体中进行了研究。所有这些启动子嵌合体都没有产生可检测的基础启动子活性。然而,每当用P(pA)-BRE元件替换P(cA)-BRE时,就会观察到cGvpE介导的激活增强。含有P(pA)-BRE加上上游5(或更多)个碱基对的启动子嵌合体也通过异源pGvpE和mcGvpE蛋白获得了激活。通过对P(mcA)-BRE上游45个碱基对区域进行4个碱基的扫描诱变,确定了GvpE介导激活所需的序列。这些改变均未影响基础启动子活性,但序列TGAAACGG-n4-TGAACCAA对GvpE介导的P(mcA)激活很重要。

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