Structural dynamics of RNAP-promoter complex in late transcription initiation

项目总结： 细菌转录启动和启动子逃逸是基因表达受高度调控的早期步骤。一个 当新生RNA的5‘端与启动子的3.2区域发生冲突时，发生关键的起始步骤 特异性因子σ70(RNAR3.2)阻断σ出口通道。然后，或者将被遮挡的通道清除为 通过RNA出口通道和RNAP促进RNA正向易位以逃避启动子，或 新生RNA反向易位到NTP进入通道，导致其流产释放。我们最近做了 显示，在长时间暂停的回溯中间过程中，一小部分RNAP得到稳定 入会仪式。我们还表明，即使在新生σR3.2从RNA出口通道中移除之后 转录，转录动力学仍然比预期的延长慢。因此，我们假设一个 额外的启动子逃逸中间体进一步减缓了从起始到延伸的转变，并且 这两个中间体都扮演着监管角色。在目标1.A中，我们将阐明转录起始的结构 通过使用多个实验得出的分子内距离作为空间约束，在这些状态下进行复合 在粗粒度模拟上。在目标1.B中，我们将定义控制丰度的分子决定因素 这些后期入会的中间体。具体地说，我们将研究σ70区域的顺序和顺序 在不同启动子的启动子逃逸过程中从RNA出口通道中移除。在目标1.B中，我们 假设：(1)σR3和σR4在启动子逃逸过程中的置换遵循两步过程；(2) 在转录泡的皱缩的dna模板链中形成的凸起有助于去除这些σ RNA区域通过投射到通道中而退出通道。我们最近发现，过度的 RNAP的数量在体内许多基因的启动子处停滞不前，这些基因对应激反应和 在∆GREA/∆GREB大肠杆菌菌株(未发表)中，这种失速在高渗透条件下得到了增强。 在目标2中，我们将测试启动过程中的停顿是否发生在活细菌中，并作为调节媒介 用来应对压力。我们将通过高分辨率(1-2 nT)染色体DNA作图来验证这一假设。 活体足迹技术。我们还将建立体内smFRET转录泡大小的检测方法来测试 是否通过与目标1中研究的机制类似的机制在细菌细胞中发生起始暂停。 该项目将极大地推进转录领域，原因如下：(1)抗生素耐药性 是一个严重的公共卫生问题。阐明细菌基因调控的机制对于 开发有效的抗菌疗法；(2)保护RNAP结构的许多特征 从细菌到人类的功能有助于真核酶转录机制的建模；(3) 启动过程中暂停回溯的RNAP的结构尚未确定。因此，描绘出 对于不同启动子来说，阻断σ退出通道的RNA70区域的空间重排将提供有价值的 对启动子逃逸机制的洞察。

PROJECT SUMMARY: Bacterial transcription initiation and promoter escape are highly-regulated early steps of gene expression. A critical initiation step occurs when the 5'-end of the nascent RNA clashes with region 3.2 of the promoter specificity factor σ70 (σR3.2) occluding the RNA exit channel. Then, either the occluded channel is cleared to facilitate RNA forward translocation through the RNA exit channel and RNAP to escape the promoter, or the nascent RNA back-translocates into the NTP entry channel, leading to its abortive release. We have recently shown that a fraction of RNAPs get stabilized in a long-lived paused backtracked intermediate during initiation. We have also shown that even after removal of σR3.2 from the RNA exit channel by the nascent transcript, transcription kinetics is still slower than expected for elongation. Therefore, we hypothesize an additional promoter escape-intermediate further slows down the transition from initiation to elongation, and that both intermediates have regulatory roles. In Aim 1.A, we will elucidate the structures of the transcription initiation complex in these states by using multiple experimentally-derived intramolecular distances as spatial constraints on coarse-grained simulations. In Aim 1.B, we will define the molecular determinants controlling the abundance of these late initiation intermediates. Specifically, we will examine the sequence and order in which σ70 regions are removed from the RNA exit channel during promoter escape for different promoters. In Aim 1.B we hypothesize that: (1) displacement of σR3 & σR4 during promoter escape follows a two-step process; (2) the bulge formed in the scrunched DNA template strand of the transcription bubble assists in removal of these σ regions from the RNA exit channel by projecting into the channel. We recently discovered that an excessive number of RNAPs stall at promoters of many genes in vivo that are essential for stress-response and that stalling is enhanced under hyperosmotic conditions in a ∆greA/∆greB E. coli strain (unpublished). In Aim 2 we will test whether pausing in initiation occurs in live bacteria and serves as a regulatory intermediate for stress response. We will test this hypothesis by high-resolution (1-2 nt) chromosomal DNA mapping & footprinting in vivo techniques. We will also develop in vivo smFRET transcription bubble size assay to test whether pausing in initiation occurs in the bacterial cell through a mechanism similar to that studied in Aim 1. This project will significantly advance the field of transcription for the following reasons: (1) antibiotic resistance is a serious public health concern. Elucidating the mechanisms of bacterial gene regulation is crucial for the development of effective antimicrobial therapy; (2) the conservation of many features of RNAP structure & function from bacteria to humans facilitates modeling of transcription mechanisms for eukaryotic enzymes; (3) the structure of paused-backtracked RNAP in initiation has not yet been determined. Therefore, delineating the spatial rearrangements of σ70 regions blocking the RNA exit channel for different promoters will provide valuable insight into the mechanism of promoter escape.