Prosseda Gianni, Falconi Maurizio, Giangrossi Mara, Gualerzi Claudio O, Micheli Gioacchino, Colonna Bianca
Dip. Biologia Cellulare e dello Sviluppo, University La Sapienza, 00185 Roma, Italy.
Mol Microbiol. 2004 Jan;51(2):523-37. doi: 10.1046/j.1365-2958.2003.03848.x.
In the human enteropathogen Shigella transcription of virF, the primary regulator of the invasion functions, is strictly temperature-dependent and is antagonistically mediated by H-NS and FIS, which bind to specific sites on the virF promoter. Here we report on the relevance of DNA geometry to the thermoregulation of virF and demonstrate that the virF promoter hosts a major DNA bend halfway between two H-NS sites. The bent region has been mutagenized in vitro to mimic temperature-induced changes of DNA curvature. Functional analysis of curvature mutants and of promoter constructs in which the two H-NS sites are phased-out by a half-helix turn reveals that modifying the spatial relationships between these sites severely affects the interaction of H-NS with the virF promoter, as well as its in vivo and in vitro temperature-dependent activity. The role of promoter curvature as thermosensor is also compatible with the present observation that, with increasing temperature, the virF bending centre moves downstream at a rate having its maximum around the transition temperature, abruptly unmasking a binding site for the transcriptional activator FIS.
在人类肠道致病菌志贺氏菌中，侵袭功能的主要调节因子virF的转录严格依赖温度，且由结合在virF启动子特定位点上的H-NS和FIS进行拮抗介导。在此，我们报告了DNA几何形状与virF温度调节的相关性，并证明virF启动子在两个H-NS位点之间的中点处存在一个主要的DNA弯曲。已在体外对弯曲区域进行诱变，以模拟温度诱导的DNA曲率变化。对曲率突变体以及通过半个螺旋转角使两个H-NS位点相位错开的启动子构建体进行功能分析，结果表明，改变这些位点之间的空间关系会严重影响H-NS与virF启动子的相互作用，以及其体内和体外的温度依赖性活性。启动子曲率作为温度传感器的作用也与目前的观察结果相符，即随着温度升高，virF弯曲中心以在转变温度附近达到最大值的速率向下游移动，从而突然暴露转录激活因子FIS的结合位点。
