Hillebrand Annette, Wurm Reinhild, Menzel Artur, Wagner Rolf
Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany.
Biol Chem. 2005 Jun;386(6):523-34. doi: 10.1515/BC.2005.062.
Ribosomal RNAs in E. coli are transcribed from seven operons, which are highly conserved in their organization and sequence. However, the upstream regulatory DNA regions differ considerably, suggesting differences in regulation. We have therefore analyzed the conformation of all seven DNA elements located upstream of the major E. coli rRNA P1 promoters. As judged by temperature-dependent gel electrophoresis with isolated DNA fragments comprising the individual P1 promoters and the complete upstream regulatory regions, all seven rRNA upstream sequences are intrinsically curved. The degree of intrinsic curvature was highest for the rrnB and rrnD fragments and less pronounced for the rrnA and rrnE operons. Comparison of the experimentally determined differences in curvature with programs for the prediction of DNA conformation revealed a generally high degree of conformity. Moreover, the analysis showed that the center of curvature is located at about the same position in all fragments. The different upstream regions were analyzed for their capacity to bind the transcription factors FIS and H-NS, which are known as antagonists in the regulation of rRNA synthesis. Gel retardation experiments revealed that both proteins interact with the upstream promoter regions of all seven rDNA fragments, with the affinities of the different DNA fragments for FIS and H-NS and the structure of the resulting complexes deviating considerably. FIS binding was non-cooperative, and at comparable protein concentrations the occupancy of the different DNA fragments varied between two and four binding sites. In contrast, H-NS was shown to bind cooperatively and intermediate states of occupancy could not be resolved for each fragment. The different gel electrophoretic mobilities of the individual DNA/protein complexes indicate variable structures and topologies of the upstream activating sequence regulatory complexes. Our results are highly suggestive of differential regulation of the individual rRNA operons.
大肠杆菌中的核糖体RNA由7个操纵子转录而来，这些操纵子在组织和序列上高度保守。然而，上游调控DNA区域差异很大，表明调控存在差异。因此，我们分析了位于大肠杆菌主要rRNA P1启动子上游的所有7个DNA元件的构象。通过对包含各个P1启动子和完整上游调控区域的分离DNA片段进行温度依赖性凝胶电泳判断，所有7个rRNA上游序列都具有固有弯曲。rrnB和rrnD片段的固有弯曲程度最高，而rrnA和rrnE操纵子的弯曲程度则不太明显。将实验测定的曲率差异与DNA构象预测程序进行比较，结果显示总体一致性较高。此外，分析表明所有片段的曲率中心位置大致相同。我们分析了不同上游区域结合转录因子FIS和H-NS的能力，这两种转录因子在rRNA合成调控中是拮抗剂。凝胶阻滞实验表明，这两种蛋白质都与所有7个rDNA片段的上游启动子区域相互作用，不同DNA片段对FIS和H-NS的亲和力以及形成的复合物结构差异很大。FIS的结合是非协同性的，在相当的蛋白质浓度下，不同DNA片段的结合位点占有率在2到4个之间变化。相比之下，H-NS显示出协同结合，每个片段的占据中间状态无法分辨。各个DNA/蛋白质复合物不同的凝胶电泳迁移率表明上游激活序列调控复合物的结构和拓扑结构各不相同。我们的结果强烈暗示了各个rRNA操纵子存在差异调控。
