Interaction of the atypical prokaryotic transcription activator FlhD2C2 with early promoters of the flagellar gene hierarchy
Interaction of the atypical prokaryotic transcription activator FlhD2C2 with early promoters of the flagellar gene hierarchy
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DOI：
10.1016/s0022-2836(02)00600-9
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发表时间：
2002-08-09
影响因子：
5.6
通讯作者：
Hughes, C
中科院分区：
文献类型：
--
作者：
Claret, L;Hughes, C
转录激活因子FlhD（2）C（2）是细菌鞭毛生物发生和群集迁移的主要调节因子，激活大鞭毛基因层级的“早期”II类启动子。使用引物延伸，带移测定，酶和化学足迹法，我们描述了FlhD（2）C（2）异源四聚体的四个II类鞭毛操纵子，fliAZ，flhBA和不同的flgAMN和flgBCD（EFGHIJ）的启动子区的结合。每个启动子区由单个异源四聚体结合，即flgAMN和flgBCD操纵子的特征在于单个FlhD（2）C（2）结合位点。结合亲和力不同，并与先前报道的启动子强度和激活顺序。甲基化保护和干扰以及脱嘌呤和脱嘧啶干扰提供了与启动子-35序列重叠的常见46-59 bp DNA酶I足迹内关键碱基的详细图谱。这些数据和12个已知的大肠杆菌、奇异变形杆菌和鼠伤寒沙门氏菌的II类启动子序列的汇编允许确定具有假对称性的FlhD（2）C（2）结合位点，其包含两个17-18 bp反向重复序列，每个都是共有的FlhD（2）C（2）盒，由10-11 bp间隔区隔开。DNaseI超敏反应表明，结合可能会导致启动子区域的构象变化。只有FlhC亚基可以独立地与DNA结合，但FlhD增强了相互作用的特异性和稳定性。在此，光交联确定FlhC和稳定化FlhD都接触FlhD（2）C（2）四聚体内的DNA。我们的数据表明，FlhD（2）C（2）/DNA复合物的识别特异性和稳定性需要蛋白质-蛋白质相互作用以及FlhC和FlhD亚基与DNA的相互作用。FlhD（2）C（2）/DNA复合物中FlhD和FlhC亚基的这些特征在原核调节子中是非常典型的。(C)2002爱思唯尔科技有限公司。保留所有权利。
The transcriptional activator FlhD(2)C(2) is the master regulator of bacterial flagellum biogenesis and swarming migration, activating the "early" class II promoters of the large flagellar gene hierarchy. Using primer extensions, band-shift assays, and enzymatic and chemical footprinting, we describe the binding of the FlhD(2)C(2) heterotetramer to the promoter regions of four class II flagella operons, fliAZ, flhBA and the divergent flgAMN and flgBCD(EFGHIJ). Each of the promoter regions was bound by a single heterotetramer, i.e. the flgAMN and flgBCD operons are characterised by a single FlhD(2)C(2) binding site. Binding affinity differed, and correlated with previously reported promoter strength and order of activation. Methylation protection and interference, and depurination and depyrimidation interference provided a detailed map of critical bases within a common 46-59 bp DNaseI footprint overlapping the promoter -35 sequences. These data and compilation of the 12 known class II promoter sequences of Escherichia coli, Proteus mirabilis and Salmonella typhimurium allowed determination of a FlhD(2)C(2) binding site with pseudo symmetry, comprising two 17-18 bp inverted repeats, each a consensus FlhD(2)C(2) box, separated by a 10-11 bp spacer. DNaseI hypersensitivity indicated that binding may cause a conformational change in the promoter regions. Only the FlhC subunit can bind DNA independently, but the specificity and stability of the interaction is strengthened by FlhD. Here, photo-crosslinking established that both FlhC and the stabilising FlhD contact the DNA within the FlhD(2)C(2) tetramer. Our data suggest that specificity of recognition and stability of the FlhD(2)C(2)/DNA complex require protein-protein interaction and interaction of both FlhC and FlhD subunits with DNA. These characteristics of the FlhD and FlhC subunits in the FlhD(2)C(2)/DNA complex are strikingly atypical of prokaryotic regulators. (C) 2002 Elsevier Science Ltd. All rights reserved.