Grainger David C, Belyaeva Tamara A, Lee David J, Hyde Eva I, Busby Stephen J W
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Mol Microbiol. 2003 Apr;48(2):335-48. doi: 10.1046/j.1365-2958.2003.t01-1-03434.x.
The Escherichia coli MelR protein is a melibiose-triggered transcription factor, belonging to the AraC family, that activates transcription initiation at the melAB promoter. Activation is dependent on the binding of MelR to four 18 bp sites, centred at position -42.5 (site 2'), position -62.5 (site 2), position -100.5 (site 1) and position -120.5 (site 1') relative to the melAB transcription start point. Activation also depends on the binding of CRP to a single site located between MelR binding site 1 and site 2. All members of the AraC family contain two helix-turn-helix (HTH) motifs that contact two segments of the DNA major groove at target sites on the same DNA face. In this work, we have studied the binding of MelR to different sites at the melAB promoter, focusing on the orientation of binding of the two MelR HTH motifs, and the juxtaposition of the different bound MelR subunits with respect to each other. To do this, MelR was engineered to contain a single cysteine residue adjacent to either one or the other HTH motif. The MelR derivatives were purified, and the cysteine residues were tagged with p-bromoacetamidobenzyl-EDTA-Fe, an inorganic DNA cleavage reagent. Patterns of DNA cleavage after MelR binding were then used to determine the positions of the two HTH motifs at target sites. In order to simplify our analysis, we exploited an engineered derivative of the melAB promoter in which MelR binding to site 2 and site 2', in the absence of CRP, is sufficient for transcription activation. To assist in the interpretation of our results, we also used a shortened derivative of MelR, MelR173, that is able to bind to site 2 but not to site 2'. Our results show that MelR binds as a direct repeat to site 2 and site 2' with the C-terminal HTH located towards the promoter-proximal end of each site. The orientation in which MelR binds to site 2' appears to be determined by MelR-MelR interactions rather than by MelR-DNA interactions. In complementary experiments, we used genetic analysis to investigate the importance of different residues in the two HTH motifs of MelR. Epistasis experiments provided evidence that supports the proposed orientation of binding of MelR at its target site.
大肠杆菌MelR蛋白是一种由蜜二糖触发的转录因子，属于AraC家族，可激活melAB启动子处的转录起始。激活作用依赖于MelR与四个18 bp位点的结合，这些位点以相对于melAB转录起始点的-42.5位置（位点2'）、-62.5位置（位点2）、-100.5位置（位点1）和-120.5位置（位点1'）为中心。激活作用还取决于CRP与位于MelR结合位点1和位点2之间的单个位点的结合。AraC家族的所有成员都包含两个螺旋-转角-螺旋（HTH）基序，它们在同一DNA面上的靶位点与DNA大沟的两个片段接触。在这项工作中，我们研究了MelR与melAB启动子不同位点的结合，重点关注两个MelR HTH基序的结合方向，以及不同结合的MelR亚基彼此之间的并列情况。为此，对MelR进行工程改造，使其在其中一个或另一个HTH基序附近包含一个半胱氨酸残基。纯化MelR衍生物，并用无机DNA切割试剂对溴乙酰氨基苄基-EDTA-Fe标记半胱氨酸残基。然后利用MelR结合后的DNA切割模式来确定两个HTH基序在靶位点的位置。为了简化我们的分析，我们利用了melAB启动子的一种工程衍生物，在没有CRP的情况下，MelR与位点2和位点2'的结合足以激活转录。为了辅助解释我们的结果，我们还使用了MelR的一种缩短衍生物MelR173，它能够结合到位点2，但不能结合到位点2'。我们的结果表明，MelR以直接重复的形式结合到位点2和位点2'，其C端HTH朝向每个位点的启动子近端。MelR结合到位点2'的方向似乎是由MelR-MelR相互作用而非MelR-DNA相互作用决定的。在补充实验中，我们使用遗传分析来研究MelR的两个HTH基序中不同残基的重要性。上位性实验提供了支持所提出的MelR在其靶位点结合方向的证据。
