Transcription activation by FNR: Evidence for a functional activating region 2
Transcription activation by FNR: Evidence for a functional activating region 2
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DOI：
10.1128/jb.184.21.5855-5861.2002
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发表时间：
2002-11-01
影响因子：
3.2
通讯作者：
Green, J
中科院分区：
文献类型：
--
作者：
Blake, T;Barnard, A;Green, J
大肠杆菌的FNR蛋白通过组装-拆卸氧不稳定的铁硫簇来控制靶基因的转录以响应缺氧。先前的工作确定了FNR表面暴露的氨基酸（分别指定为激活区1和3 [AR 1和AR 3]）的补丁，使其能够与RNA聚合酶（RNAP）通信，从而激活转录。以前认为FNR缺乏功能性激活区2（AR 2），尽管选择补偿缺陷AR 1或不协调的铁硫簇的突变可以重新激活AR 2。在这里，我们表明FNR的两个表面暴露的赖氨酸残基（Lys 49和Lys 50）的取代损害了来自H类（FNR框中心在-41.5）而不是I类（FNR框中心在-71.5）FNR依赖性启动子的转录。当带负电荷的残基（Glu）取代Lys 49或Lys 50时，损害的程度比不带电荷的氨基酸Ala被取代时更大。定向的异源二聚体用于显示只有FNR二聚体的下游亚基受11类启动子处的Lys → Ala取代的影响。在RNAP α亚基的N-末端结构域内与环AMP受体蛋白的带正电荷的AR 2相互作用的带负电荷的补丁（（EEDE 165）-E-162）的定点诱变表明FNR的Lys 49和Lys 50与RNAP α亚基的该区域相互作用。因此，提示Lys 49和Lys 50形成FNR中功能性AR 2的一部分。
The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia via the assembly-disassembly of oxygen-labile iron-sulfur clusters. Previous work identified patches of surface-exposed amino acids (designated activating regions 1 and 3 [AR1 and AR3, respectively]) of FNR which allow it to communicate with RNA polymerase (RNAP) and thereby activate transcription. Previously it was thought that FNR lacks a functional activating region 2 (AR2), although selecting for mutations that compensate for defective AR1 or a miscoordinated iron-sulfur cluster can reactivate AR2. Here we show that the substitution of two surface-exposed lysine residues (Lys49 and Lys50) of FNR impaired transcription from class H (FNR box centered at -41.5) but not class I (FNR box centered at -71.5) FNR-dependent promoters. The degree of impairment was greater when a negatively charged residue (Glu) replaced either Lys49 or Lys50 than when uncharged amino acid Ala was substituted. Oriented heterodimers were used to show that only the downstream subunit of the FNR dimer was affected by the Lys-->Ala substitutions at a class 11 promoter. Site-directed mutagenesis of a negatively charged patch ((EEDE165)-E-162) within the N-terminal domain of the RNAP alpha subunit that interacts with the positively charged AR2 of the cyclic AMP receptor protein suggested that Lys49 and Lys50 of FNR interact with this region of the alpha subunit of RNAP. Thus, it was suggested that Lys49 and Lys50 form part of a functional AR2 in FNR.