Nou X, Braaten B, Kaltenbach L, Low D A
Department of Pathology, University of Utah, Salt Lake City 84132, USA.
EMBO J. 1995 Dec 1;14(23):5785-97. doi: 10.1002/j.1460-2075.1995.tb00267.x.
Pyelonephritis-associated pili (Pap) expression in Escherichia coli is subject to a phase variation control mechanism that is regulated by the leucine-responsive regulatory protein (Lrp), PapI, and deoxyadenosine methylase (Dam). In previous work, we found that the differential Dam methylation of two target sites in pap regulatory DNA, GATC-I and GATC II, is essential for the transition between active and inactive pap transcriptional states. Here, we identify six Lrp binding sites within the pap regulatory DNA, each separated by about three helical turns. Lrp binds with highest affinity to three sites (1, 2 and 3) proximal to the papBAp promoter. A mutational analysis indicates that the binding of Lrp to sites 2 and 3 inhibits pap transcription, which is consistent with the fact that Lrp binding site 3 is located between the --35 and --10 RNA polymerase binding region of papBAp. The addition of PapI decreases the affinity of Lrp for sites 1, 2 and 3 and increases its affinity for the distal Lrp binding sites 4 and 5. Mutations within Lrp binding sites 4 and 5 shut off pap transcription, indicating that the binding of Lrp to this pap region activates pap transcription. The pap GATC-I and GATC-II methylation sites are located within Lrp binding sites 5 and 2, respectively, providing a mechanism by which Dam controls Lrp binding and Pap phase variation.
大肠杆菌中肾盂肾炎相关菌毛（Pap）的表达受一种相变控制机制的调控，该机制由亮氨酸反应调节蛋白（Lrp）、PapI和脱氧腺苷甲基化酶（Dam）调节。在之前的研究中，我们发现pap调控DNA中两个靶位点GATC-I和GATC II的差异性Dam甲基化对于pap转录活性状态和非活性状态之间的转变至关重要。在此，我们在pap调控DNA中鉴定出6个Lrp结合位点，每个位点相隔约三个螺旋圈。Lrp与papBAp启动子近端的三个位点（1、2和3）结合亲和力最高。突变分析表明，Lrp与位点2和3的结合会抑制pap转录，这与Lrp结合位点3位于papBAp的–35和–10 RNA聚合酶结合区域之间这一事实相符。添加PapI会降低Lrp对位点1、2和3的亲和力，并增加其对远端Lrp结合位点4和5的亲和力。Lrp结合位点4和5内的突变会关闭pap转录，这表明Lrp与该pap区域的结合会激活pap转录。pap GATC-I和GATC-II甲基化位点分别位于Lrp结合位点5和2内，这提供了一种Dam控制Lrp结合和Pap相变的机制。
