Schröder O, Wagner R
Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, Düsseldorf, D-40225, Germany.
J Mol Biol. 2000 May 19;298(5):737-48. doi: 10.1006/jmbi.2000.3708.
The interaction of the bacterial regulatory protein H-NS with RNA polymerase and the ribosomal RNA P1 promoter was analyzed to better understand the mechanism of H-NS-dependent transcriptional repression. We could show that initial binding of RNA polymerase to the promoter was not inhibited by the simultaneous interaction of H-NS, although H-NS binding sites extend into the core promoter region. Binding of sigma(70)-saturated RNA polymerase and H-NS to the promoter DNA occurs cooperatively and results in a stable complex of slower gel electrophoretic mobility as compared to complexes formed with the single proteins. The presence of the upstream curved H-NS binding site contributes strongly to the cooperative RNA polymerase-promoter interaction. By KMnO(4) modification of single-stranded template nucleotides we could show that open complex formation at the rrnB P1 promoter was not inhibited by H-NS binding. An increased KMnO(4) reactivity of several positions within the open complex rather supports the view that open complex formation is stimulated in presence of H-NS. Moreover, subtle changes in the modification pattern indicate that the open complex formed in the presence of H-NS are structurally distinct from the H-NS-free complex. In vitro transcriptional analysis of the abortive and productive yields revealed that the formation of transcription products longer than three nucleotides is dramatically reduced in the presence of H-NS, while the amount of shorter abortive products remained unaffected. Together the results demonstrate that H-NS inhibits transcription at the rrnB P1 promoter not by interfering with initial RNA polymerase binding but by blocking chain elongation steps subsequent to the first (two) phosphodiester bond formations. The mechanism of H-NS dependent repression at rRNA promoters can thus be explained as a trap which inhibits substrate NTP incorporation beyond template position +3 into the initial transcribing complex.
为了更好地理解H-NS依赖性转录抑制的机制，我们分析了细菌调节蛋白H-NS与RNA聚合酶和核糖体RNA P1启动子之间的相互作用。我们发现，尽管H-NS结合位点延伸到核心启动子区域，但RNA聚合酶与启动子的初始结合并未受到H-NS同时相互作用的抑制。σ(70)饱和的RNA聚合酶和H-NS与启动子DNA的结合是协同发生的，与单个蛋白质形成的复合物相比，形成的复合物具有较慢的凝胶电泳迁移率，且更为稳定。上游弯曲的H-NS结合位点的存在对RNA聚合酶与启动子的协同相互作用有很大贡献。通过对单链模板核苷酸进行高锰酸钾修饰，我们发现rrnB P1启动子处开放复合物的形成不受H-NS结合的抑制。开放复合物中几个位置的高锰酸钾反应性增加，反而支持了在H-NS存在下开放复合物形成受到刺激的观点。此外，修饰模式的细微变化表明，在H-NS存在下形成的开放复合物在结构上与无H-NS的复合物不同。对流产和有效产量的体外转录分析表明，在H-NS存在下，长度超过三个核苷酸的转录产物的形成显著减少，而较短的流产产物的数量不受影响。这些结果共同表明，H-NS在rrnB P1启动子处抑制转录，不是通过干扰RNA聚合酶的初始结合，而是通过阻断第一个（两个）磷酸二酯键形成后的链延伸步骤。因此，rRNA启动子处H-NS依赖性抑制的机制可以解释为一种陷阱，它抑制底物NTP掺入到初始转录复合物中模板位置+3以外的位置。
