Stoyanov J V, Hobman J L, Brown N L
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Mol Microbiol. 2001 Jan;39(2):502-11. doi: 10.1046/j.1365-2958.2001.02264.x.
We have shown that the open reading frame ybbI in the genomic sequence of Escherichia coli K-12 encodes the regulator of expression of the copper-exporting ATPase, CopA. In vivo studies showed that ybbI (designated cueR for copper export regulator gene) was required for copper tolerance during growth, that disruption of cueR caused loss of copA expression and that copA gene expression was regulated by cueR and by copper or silver ions. Expression of a lacZ reporter gene under the control of the copA promoter was approximately proportional to the concentration of cupric ions in the medium, but increased more rapidly in response to silver ion concentrations. The start of the copA transcript was located by primer extension mapping, and DNase I protection assays showed that the CueR protein binds in vitro to a dyad symmetrical sequence within a 19 bp spacer sequence in the copA promoter. CueR binding occurs in vitro in both the presence and the absence of RNA polymerase with or without copper ions present but, in the presence of CueR, RNA polymerase and copper ions, permanganate-sensitive transcription complexes were formed. CueR is predicted to have an N-terminal helix-turn-helix sequence and shows similarity to MerR family regulators.
我们已经证明,大肠杆菌K-12基因组序列中的开放阅读框ybbI编码铜输出ATP酶CopA的表达调节因子。体内研究表明,ybbI(指定为铜输出调节基因cueR)在生长过程中对铜耐受性是必需的,cueR的破坏导致copA表达丧失,并且copA基因表达受cueR以及铜或银离子的调节。在copA启动子控制下的lacZ报告基因的表达与培养基中铜离子的浓度大致成比例,但对银离子浓度的反应增加得更快。通过引物延伸图谱确定了copA转录本的起始位置,DNase I保护试验表明,CueR蛋白在体外与copA启动子中19 bp间隔序列内的二元对称序列结合。无论有无RNA聚合酶以及有无铜离子存在,CueR在体外均能结合,但在存在CueR、RNA聚合酶和铜离子的情况下,会形成对高锰酸盐敏感的转录复合物。预计CueR具有N端螺旋-转角-螺旋序列,并且与MerR家族调节因子相似。