Hancock Stephen P, Stella Stefano, Cascio Duilio, Johnson Reid C
Department of Biological Chemistry, David Geffen School of Medicine at the University of California at Los Angeles, Los Angeles, California, United States of America.
Department of Energy Institute of Genomics and Proteomics, University of California at Los Angeles, Los Angeles, California, United States of America.
PLoS One. 2016 Mar 9;11(3):e0150189. doi: 10.1371/journal.pone.0150189. eCollection 2016.
The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. The affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.
丰富的Fis类核蛋白以高亲和力选择性结合关系不大的DNA序列，以调节多种DNA反应。Fis主要通过与DNA主链接触来结合DNA，并通过读取DNA序列的构象特性（最显著的是内在小沟宽度）来选择靶位点。高亲和力结合需要Fis稳定的DNA构象变化，这种变化因DNA序列而异。为了更好地理解高亲和力位点识别的分子基础，我们分析了核心Fis结合位点内部及侧翼的DNA序列对结合亲和力和DNA结构的影响。在结合位点的非接触中心包含可变序列或在大沟界面内存在变异的Fis-DNA复合物的X射线晶体结构表明，DNA可以不对称地适应Fis二聚体表面。我们表明，大沟界面内嘧啶-嘌呤碱基步移的存在和位置会影响局部DNA弯曲和小沟压缩，从而调节Fis-DNA复合物的亲和力和寿命。核心结合位点侧翼的序列也会调节复合物的亲和力、寿命以及局部和全局Fis诱导的DNA弯曲程度。特别是，15 bp核心序列上游紧邻的一个G会抑制结合和弯曲，而侧翼碱基对内的A序列会增加复合物寿命和全局DNA曲率。综上所述，我们的观察结果支持一种经过修订的DNA基序，该基序指定了高亲和力的Fis结合，并突出了Fis结合的DNA可以采用的构象范围。单个Fis-DNA复合物的亲和力和DNA构象可能是根据其特定背景的生物学功能量身定制的。
