Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.
Genome Res. 2022 May;32(5):878-892. doi: 10.1101/gr.276544.121. Epub 2022 Apr 28.
When detected at single-base-pair resolution, the genome-wide location, occupancy level, and structural organization of DNA-binding proteins provide mechanistic insights into genome regulation. Here we use ChIP-exo to provide a near-base-pair resolution view of the epigenomic organization of the transcription machinery and nucleoid structural proteins at the time when cells are growing exponentially and upon rapid reprogramming (acute heat shock). We examined the site specificity of three sigma factors (RpoD/σ, RpoH/σ, and RpoN/σ), RNA polymerase (RNAP or RpoA, -B, -C), and two nucleoid proteins (Fis and IHF). We suggest that DNA shape at the flanks of cognate motifs helps drive site specificity. We find that although RNAP and sigma factors occupy active cognate promoters, RpoH and RpoN can occupy quiescent promoters without the presence of RNAP. Thus, promoter-bound sigma factors can be triggered to recruit RNAP by a mechanism that is distinct from an obligatory cycle of free sigma binding RNAP followed by promoter binding. These findings add new dimensions to how sigma factors achieve promoter specificity through DNA sequence and shape, and further define mechanistic steps in regulated genome-wide assembly of RNAP at promoters in .
当以单碱基分辨率检测时，DNA 结合蛋白的全基因组位置、占据水平和结构组织为基因组调控提供了机制上的见解。在这里，我们使用 ChIP-exo 在细胞快速增殖和快速重编程（急性热休克）时，提供转录机器和核区结构蛋白的表观基因组组织的近碱基分辨率视图。我们检查了三个 sigma 因子（RpoD/σ、RpoH/σ 和 RpoN/σ）、RNA 聚合酶（RNAP 或 RpoA、-B、-C）和两个核区蛋白（Fis 和 IHF）的位点特异性。我们认为，同源基序侧翼的 DNA 形状有助于驱动位点特异性。我们发现，尽管 RNAP 和 sigma 因子占据活性同源启动子，但 RpoH 和 RpoN 可以在没有 RNAP 的情况下占据静止启动子。因此，通过一种不同于游离 sigma 结合 RNAP 随后启动子结合的强制性循环的机制，启动子结合的 sigma 因子可以被触发招募 RNAP。这些发现为 sigma 因子如何通过 DNA 序列和形状实现启动子特异性增加了新的维度，并进一步定义了在 中受调控的全基因组组装 RNAP 到启动子的机制步骤。
