Gosink K K, Ross W, Leirmo S, Osuna R, Finkel S E, Johnson R C, Gourse R L
Department of Bacteriology, University of Wisconsin, Madison 53706.
J Bacteriol. 1993 Mar;175(6):1580-9. doi: 10.1128/jb.175.6.1580-1589.1993.
The Escherichia coli Fis protein binds to three sites in the upstream activation region of the rrnB P1 promoter and enhances transcription 5- to 10-fold in vivo. In this report, we investigate the mechanism of Fis-dependent activation of transcription. We show that stimulation of rrnB P1 transcription by Fis can occur on linear DNA templates and does not require DNA upstream of the promoter-proximal Fis site I. Mutants of Fis defective for Hin-mediated recombination have been isolated previously and have defined an N-terminal domain required for DNA inversion by Hin in addition to the C-terminal domain which is required for DNA binding. Several of these mutants were found to be defective in stimulation of rrnB P1 transcription in vivo and in vitro. Activation-defective mutants fall into three classes: those that fail to bind to the upstream activation region, those that bind but fail to bend the DNA normally, and those that bind and bend but still fail to activate transcription. We conclude that it is unlikely that Fis functions by simply bringing upstream sequences or bound factors into the proximity of RNA polymerase to activate transcription. Rather, the data are most easily interpreted in terms of transcription activation by direct interactions between Fis and RNA polymerase, requiring precise positioning of the two proteins facilitated by bending of the DNA binding site.
大肠杆菌Fis蛋白与rrnB P1启动子上游激活区域的三个位点结合，并在体内将转录增强5至10倍。在本报告中，我们研究了Fis依赖的转录激活机制。我们发现，Fis对rrnB P1转录的刺激可在线性DNA模板上发生，且不需要启动子近端Fis位点I上游的DNA。先前已分离出对Hin介导的重组有缺陷的Fis突变体，除了DNA结合所需的C末端结构域外，还定义了Hin进行DNA倒位所需的N末端结构域。发现其中几个突变体在体内和体外对rrnB P1转录的刺激均有缺陷。激活缺陷型突变体分为三类：那些无法与上游激活区域结合的突变体，那些能够结合但无法正常弯曲DNA的突变体，以及那些能够结合并弯曲但仍无法激活转录的突变体。我们得出结论，Fis不太可能通过简单地将上游序列或结合因子带到RNA聚合酶附近来激活转录。相反，这些数据最容易通过Fis与RNA聚合酶之间的直接相互作用来解释转录激活，这需要通过DNA结合位点的弯曲来促进两种蛋白质的精确定位。
