Multiple physiological roles of both specific and non-specific DNA binding modes of HU protein in Escherichia coli. Multiple physiological roles of both specific and non-specific DNA binding modes of HU protein in Escherichia coli

The histone-like protein HU is crucial for genome organization and the expression of many genes in Escherichia coli. HU binds DNA independent of any specific nucleotide sequence but exhibits two binding affinities; low-affinity to any B-DNA (non-specific) and high affinity to DNA with distortions like kinks and cruciforms (structure-specific). We validated and defined the three conserved lysine residues, K3, K18, and K83, in HU as critical amino acid residues for the non-specific binding and the conserved P63 together with the lysine residues critical for the structure-specific binding both in vitro and in vivo. By determining the effects of disrupting the two DNA binding modes on various HU-dependent physiological processes, we demonstrate that the DNA structure-specific binding regulates expression of many genes including those involved in DNA metabolic processes that control chromosome maintenance and partition. On the other hand, HU associates with the chromosome at numerous sites primarily through the lysines-mediated non-specific binding and control chromosome dynamics and structural maintenance. Thus, we demonstrate that two different DNA binding mode of HU plays separate roles: (i) The high-affinity DNA structure-specific binding regulates many distinct DNA metabolic processes, primarily through transcription regulation. (ii) Its low-affinity, non-specific binding directly helps general organization of the genome. Overall design: Three biological replicates were analyzed for each strain

类组蛋白HU（histone-like protein HU）对大肠杆菌（Escherichia coli）的基因组组织以及众多基因的表达至关重要。HU可与DNA结合且不依赖任何特定核苷酸序列，但展现出两种结合亲和力模式：对任意B型DNA（B-DNA）的低亲和力（非特异性结合），以及对存在扭结（kinks）、十字形结构（cruciforms）等畸变的DNA的高亲和力（结构特异性结合）。本研究通过体外（in vitro）和体内（in vivo）实验，验证并明确了HU中三个保守赖氨酸残基K3、K18和K83为非特异性结合的关键氨基酸残基，同时确定保守残基P63与对结构特异性结合至关重要的赖氨酸残基共同作为结构特异性结合的关键位点。通过探究破坏两种DNA结合模式对多种HU依赖型生理过程的影响，本研究证实：DNA结构特异性结合可调控众多基因的表达，其中涵盖参与调控染色体维持与分离的DNA代谢过程相关基因。另一方面，HU主要通过赖氨酸介导的非特异性结合在染色体的大量位点处结合，并参与调控染色体动态变化与结构维持。综上，本研究证实HU的两种不同DNA结合模式发挥着独立的功能：(i) 高亲和力的DNA结构特异性结合主要通过转录调控，调控多种不同的DNA代谢过程；(ii) 低亲和力的非特异性结合则直接协助完成基因组的整体组织。实验整体设计：每株菌株均设置3次生物学重复（biological replicates）进行分析。