大肠杆菌RNA聚合酶开放启动子复合物稳定性的结构起源

R. Saecker, James Chen, C. Chiu, B. Malone, J. Sotiris, Mark Ebrahim, L. Y. Yen, E. Eng, S. Darst
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引用次数: 18

摘要

意义调控转录起始复合物的形成速率和寿命是基因表达调控的关键。在大肠杆菌中,单核苷酸的变化可以将RNA聚合酶(RNAP)启动子DNA复合体的半衰期改变一个数量级以上。人们对这些效应的起源知之甚少。利用低温电镜,我们发现转录泡的序列或大小的微小变化会引发rna - DNA相互作用和DNA碱基堆叠的全局变化。我们的研究结果表明,非加性结构变化允许一些关键的DNA位置将转录起始复合物的寿命从几秒调整到几小时,从而影响RNA合成初始步骤的速率和效率。在所有生物体中,基因表达的第一步都需要打开DNA双链,暴露其中一条链用于模板化RNA合成。在大肠杆菌中,启动子DNA序列从根本上决定了RNA聚合酶(RNAP)形成“开放”复合物(RPo)的速度,RPo是持续数秒还是数小时,以及RNAP从起始到延伸的转变速度。这些速率控制着体内启动子的强度,但它们的结构起源在很大程度上仍然未知。在这里,我们使用低温电子显微镜来确定在37°C下三个不同寿命的启动子中重新形成的RPo的结构:λPR (t1/2 ~ 10小时),T7A1 (t1/2 ~ 4分钟)和λPR的点突变体(λPR- 5c) (t1/2 ~ 2小时)。在λPR上填充了两种不同的RPo构象,可能代表了在溶液研究中观察到的RPo的生产和非生产形式。我们发现,转录泡中DNA序列和长度的变化在起始位点上游(+1)整体上改变了DNA - rnap相互作用网络、碱基堆叠和转录泡单链DNA的链顺序;这些差异在气泡之外传播到上游和下游的DNA。在转录泡扩大一个碱基(T7A1)后,非模板链在活性位点裂缝内“卷曲”;模板链在气泡上游边缘的裂缝外凸起。这些结构说明了有限的序列变化如何触发转录泡中的全局改变,从而调节RPo寿命并影响转录周期的后续步骤。
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Structural origins of Escherichia coli RNA polymerase open promoter complex stability
Significance The modulation of the rate of formation and of the lifetime of transcription initiation complexes is a critical point in gene expression control. In Escherichia coli, single-nucleotide changes can change the half-life of an RNA polymerase (RNAP)–promoter DNA complex by more than an order of magnitude. The origins of these effects are poorly understood. Using cryoelectron microscopy, we find that small alterations in the sequence or size of the transcription bubble trigger global changes in RNAP–DNA interactions and in DNA base stacking. Our results reveal that nonadditive structural changes allow a few crucial DNA positions to tune the transcription initiation complex lifetime from seconds to hours, influencing the rate and efficiency of the initial steps of RNA synthesis. The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms “open” complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo, but their structural origins remain largely unknown. Here, we use cryoelectron microscopy to determine the structures of RPo formed de novo at three promoters with widely differing lifetimes at 37 °C: λPR (t1/2 ∼10 h), T7A1 (t1/2 ∼4 min), and a point mutant in λPR (λPR-5C) (t1/2 ∼2 h). Two distinct RPo conformers are populated at λPR, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA–RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate strand “scrunches” inside the active site cleft; the template strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate the RPo lifetime and affect the subsequent steps of the transcription cycle.
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