Binding of the Escherichia coli MelR protein to the melAB promoter:: orientation of MelR subunits and investigation of MelR-DNA contacts
Binding of the Escherichia coli MelR protein to the melAB promoter:: orientation of MelR subunits and investigation of MelR-DNA contacts
复制标题
DOI：
10.1046/j.1365-2958.2003.t01-1-03434.x
复制
发表时间：
2003-04-01
影响因子：
3.6
通讯作者：
Busby, SJW
中科院分区：
文献类型：
--
作者：
Grainger, DC;Belyaeva, TA;Busby, SJW
大肠杆菌MERR蛋白是一种由Melibiose触发的转录因子，属于AraC家族，能激活MelAB启动子的转录启动。激活依赖于MelR与4个18bp位点的结合，这些位点位于相对于MelAB转录起始点的-42.5位(2‘位)、-62.5位(2位)、-100.5位(1位)和-120.5位(1’位)。激活还依赖于C反应蛋白与位于MERR结合位点1和位点2之间的单个位点的结合。AraC家族的所有成员都含有两个螺旋-转角螺旋(HTH)基序，这两个基序在同一DNA面上的靶部位接触DNA主槽的两个片段。在这项工作中，我们研究了MelR与MelAB启动子上不同位点的结合，重点研究了两个MelR HTH基序的结合方向，以及不同结合的MelR亚基之间的并置。为了做到这一点，MALR被设计成在一个或另一个HTH基序附近含有一个半胱氨酸残基。对MALR衍生物进行纯化，半胱氨酸残基用无机DNA裂解试剂对溴乙酰氨基苯甲基-EDTA-Fe标记。然后利用MERR结合后的DNA切割模式来确定两个HTH基序在目标位置的位置。为了简化我们的分析，我们利用了MelAB启动子的一个工程衍生物，其中在没有CRP的情况下，MelR与2位点和2‘位点结合足以进行转录激活。为了帮助解释我们的结果，我们还使用了MelR的一个缩短的衍生物MelR173，它能够与第二位点结合，但不能与第二位点结合。我们的结果表明，MelR以直接重复的方式与2位点和2‘位点结合，C-末端的HTH位于每个位点的启动子近端。MALR与2‘位的结合方向似乎是由MERR-MERR相互作用决定的，而不是MERR-DNA相互作用决定的。在互补性实验中，我们使用遗传分析来研究MERR的两个HTH基序中不同残基的重要性。上位性实验提供了证据，支持MERR在其靶点结合的方向。
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.