DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation
DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation
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DOI：
10.1088/1478-3975/10/6/066005
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发表时间：
2013-12-01
期刊：
影响因子：
2
通讯作者：
Phillips, Rob
中科院分区：
文献类型：
--
作者：
Boedicker, James Q.;Garcia, Hernan G.;Phillips, Rob
作为生命的主要信息分子，DNA受到广泛的物理操纵。使双螺旋DNA变形所需的能量取决于序列，这种DNA的机械密码影响基因调节，例如通过核小体定位。在这里，我们检查了DNA在细菌转录因子介导的环路中依赖于序列的灵活性，在这种情况下，序列的作用仍然知之甚少。使用一套由Lac阻遏物抑制的合成结构和两个在体外显示出较大灵活性差异的已知序列，我们对DNA序列如何影响环的形成做出了精确的统计力学预测，并使用体内转录和体外单分子分析验证了这些预测。令人惊讶的是，序列依赖的灵活性并不影响体内的基因调控。通过理论和实验量化序列和DNA弯曲蛋白HU对DNA力学性质的相对贡献，我们揭示了HU弯曲主导DNA力学并掩盖了固有的序列依赖柔性。这种对基因组中机械调控信息如何编码的定量理解将是朝着预测单碱基对分辨率下的基因调控迈出的关键一步。
As the chief informational molecule of life, DNA is subject to extensive physical manipulations. The energy required to deform double-helical DNA depends on sequence, and this mechanical code of DNA influences gene regulation, such as through nucleosome positioning. Here we examine the sequence-dependent flexibility of DNA in bacterial transcription factor-mediated looping, a context for which the role of sequence remains poorly understood. Using a suite of synthetic constructs repressed by the Lac repressor and two well-known sequences that show large flexibility differences in vitro, we make precise statistical mechanical predictions as to how DNA sequence influences loop formation and test these predictions using in vivo transcription and in vitro single-molecule assays. Surprisingly, sequence-dependent flexibility does not affect in vivo gene regulation. By theoretically and experimentally quantifying the relative contributions of sequence and the DNA-bending protein HU to DNA mechanical properties, we reveal that bending by HU dominates DNA mechanics and masks intrinsic sequence-dependent flexibility. Such a quantitative understanding of how mechanical regulatory information is encoded in the genome will be a key step towards a predictive understanding of gene regulation at single-base pair resolution.