Susanna K A, van der Werff A F, den Hengst C D, Calles B, Salas M, Venema G, Hamoen L W, Kuipers O P
Department of Genetics, University of Groningen, NL-9751 NN Haren, The Netherlands.
J Bacteriol. 2004 Feb;186(4):1120-8. doi: 10.1128/JB.186.4.1120-1128.2004.
The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK. ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machinery and of genes required for homologous recombination. In vivo and in vitro experiments have shown that ComK is responsible for transcription activation at the comG promoter. In this study, we investigated the mechanism of this transcription activation. The intrinsic binding characteristics of RNA polymerase with and without ComK at the comG promoter were determined, demonstrating that ComK stabilizes the binding of RNA polymerase to the comG promoter. This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNA resulted in an almost complete abolishment of stabilization of RNA polymerase binding. Furthermore, a strong requirement for the presence of an extra AT box in addition to the common ComK-binding site was shown. In vitro transcription with B. subtilis RNA polymerase reconstituted with wild-type alpha-subunits and with C-terminal deletion mutants of the alpha-subunits was performed, demonstrating that these deletions do not abolish transcription activation by ComK. This indicates that ComK is not a type I activator. We also show that ComK is not required for open complex formation. A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase.
枯草芽孢杆菌感受态的发育受复杂信号转导级联调控，该级联导致感受态转录因子的合成，其由comK编码。ComK是编码DNA结合和摄取机制的晚期感受态基因以及同源重组所需基因转录所必需的。体内和体外实验表明，ComK负责comG启动子处的转录激活。在本研究中，我们研究了这种转录激活的机制。测定了RNA聚合酶在有和没有ComK情况下在comG启动子处的内在结合特性，表明ComK稳定了RNA聚合酶与comG启动子的结合。这种稳定作用可能是通过与上游DNA的相互作用发生的，因为上游DNA的缺失导致RNA聚合酶结合稳定作用几乎完全消除。此外，还表明除了常见的ComK结合位点外，强烈需要额外的AT框存在。用野生型α亚基和α亚基的C末端缺失突变体重构的枯草芽孢杆菌RNA聚合酶进行体外转录，表明这些缺失不会消除ComK的转录激活作用。这表明ComK不是I型激活剂。我们还表明，开放复合物的形成不需要ComK。提出了一种转录激活的可能机制，这意味着ComK的主要刺激作用是对RNA聚合酶的结合。
