Olekhnovich I, Gussin G N
Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA.
Gene. 1998 Nov 26;223(1-2):247-55. doi: 10.1016/s0378-1119(98)00243-1.
TrpI protein, the activator of transcription of the trpBA operon of three species of fluorescent Pseudomonads, bends the DNA when it forms either of two well-characterized complexes with the trpBA regulatory region. In complex 1, TrpI is bound only to its strong binding site (site I), whereas in complex 2, which is required for activation of the trpBA promoter, TrpI is bound both to site I and to the weaker site II. Indoleglycerol phosphate (InGP) strongly stimulates formation of complex 2 and is required for activation. The present study focuses on the binding of TrpI to DNA containing a duplication of site I and the effect of the duplication on TrpI-induced DNA bending. We find that even on DNA containing a tandem (direct or inverted) duplication of site I, the formation of DNA-TrpI complexes with both sites occupied is strongly stimulated by InGP. Thus, even when TrpI binding to two adjacent sites needs not be cooperative, InGP significantly promotes the formation of complex 2. Gel binding data indicate that InGP can have several effects: (1) TrpI molecules bound to either of two adjacent strong binding sites appear to interfere with binding to the other site; InGP relieves this apparent interference. (2) InGP increases the intrinsic affinity of TrpI for sites I and II and/or enhances cooperative TrpI binding to adjacent DNA sites. Furthermore, a third molecule of TrpI can form a footprint adjacent to the duplication on DNA containing a direct (but not inverted) repeat of site I, indicating that TrpI bound to site I is oriented asymmetrically in spite of the quasi-symmetry of the binding site. The calculated bending angle for DNA in complex 2 is increased by approximately 20 degrees when site I is substituted in either orientation for site II; thus, on DNA containing a site I duplication, the bending angle of complex 2 is nearly twice that of complex 1.
色氨酸I蛋白是三种荧光假单胞菌trpBA操纵子转录的激活因子，当它与trpBA调控区域形成两种特征明确的复合物中的任何一种时，都会使DNA弯曲。在复合物1中，色氨酸I蛋白仅与它的强结合位点（位点I）结合，而在trpBA启动子激活所需的复合物2中，色氨酸I蛋白既与位点I结合，也与较弱的位点II结合。吲哚甘油磷酸（InGP）强烈刺激复合物2的形成，并且是激活所必需的。本研究重点关注色氨酸I蛋白与含有位点I重复序列的DNA的结合以及该重复对色氨酸I蛋白诱导的DNA弯曲的影响。我们发现，即使在含有位点I串联（正向或反向）重复序列的DNA上，InGP也会强烈刺激两个位点都被占据的DNA - 色氨酸I蛋白复合物的形成。因此，即使色氨酸I蛋白与两个相邻位点的结合不需要协同作用，InGP也能显著促进复合物2的形成。凝胶结合数据表明，InGP可以有多种作用：（1）与两个相邻强结合位点中的任何一个结合的色氨酸I蛋白分子似乎会干扰与另一个位点的结合；InGP消除了这种明显的干扰。（2）InGP增加了色氨酸I蛋白对位点I和位点II的内在亲和力，和/或增强了色氨酸I蛋白与相邻DNA位点的协同结合。此外，第三个色氨酸I蛋白分子可以在含有位点I正向（但不是反向）重复序列的DNA上，在重复序列附近形成一个足迹，这表明尽管结合位点具有准对称性，但与位点I结合的色氨酸I蛋白的取向是不对称的。当位点I以任何一种取向替代位点II时，复合物2中DNA的计算弯曲角度增加约20度；因此，在含有位点I重复序列的DNA上，复合物2的弯曲角度几乎是复合物1的两倍。
