Rochman Mark, Blot Nico, Dyachenko Marina, Glaser Gad, Travers Andrew, Muskhelishvili Georgi
Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, D-35043 Marburg, Germany.
Mol Microbiol. 2004 Jul;53(1):143-52. doi: 10.1111/j.1365-2958.2004.04126.x.
The stable RNA promoters of Escherichia coli are exquisitely sensitive to variations in the superhelical density of DNA. Previously, we have shown that binding of the DNA architectural protein FIS at the upstream activating sequences (UASs) of stable RNA promoters prevents the transcription complexes from inactivation induced by changes in the supercoiling level of DNA. Here, we identify a strong FIS binding site 89 bp upstream of the previously described cluster of FIS binding sites located between positions -64 and -150 in the rrnA P1 UAS. Binding of FIS to this 'far upstream sequence' allows the recruitment of additional FIS molecules to the region. We demonstrate that, upon DNA relaxation, the maintenance of promoter activity requires, in addition to UAS, the presence of the far upstream sequence. The far upstream sequence shows no effect in the absence of an intact cluster. This requirement for the integrity of the region encompassing the far upstream sequence and the UAS cluster is correlated with the in vitro modulation of binding of FIS to UAS and interaction of RNA polymerase with the UP element and the region around the transcriptional start point. Our results suggest that, at the rrnA P1 promoter, the entire region comprising the UAS and the far upstream sequence is involved in the assembly of the transcription initiation complex. We propose that the extensive engagement of upstream DNA in this nucleoprotein complex locally compensates for the lack of torsional strain in relaxed DNA, thus increasing the resistance of the promoter to global DNA relaxation.
大肠杆菌的稳定RNA启动子对DNA超螺旋密度的变化极为敏感。此前，我们已经表明，DNA结构蛋白FIS结合在稳定RNA启动子的上游激活序列（UASs）上，可防止转录复合物因DNA超螺旋水平变化而失活。在此，我们在rrnA P1 UAS中位于-64至-150位之间的先前描述的FIS结合位点簇上游89 bp处鉴定出一个强FIS结合位点。FIS与这个“远上游序列”的结合允许额外的FIS分子募集到该区域。我们证明，在DNA松弛时，启动子活性的维持除了需要UAS外，还需要远上游序列的存在。在没有完整簇的情况下，远上游序列没有影响。对包含远上游序列和UAS簇的区域完整性的这种需求与FIS与UAS结合的体外调节以及RNA聚合酶与UP元件和转录起始点周围区域的相互作用相关。我们的结果表明，在rrnA P1启动子处，包含UAS和远上游序列的整个区域参与转录起始复合物的组装。我们提出，上游DNA在这种核蛋白复合物中的广泛参与局部补偿了松弛DNA中缺乏的扭转应变，从而增加了启动子对全局DNA松弛的抗性。
