权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Structural dynamics of RNAP-promoter complex in late transcription initiation

RNAP启动子复合物在转录起始后期的结构动力学

基本信息

批准号：
10321269
负责人：
SHIMON WEISS
金额：
$ 53.08万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-02-01 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10321269
关键词：
Adopted Antibiotic Resistance Antibiotics Back Bacteria Bacterial Genes Bacterial RNA Biochemical Biological Biological Assay Biology Cells Characteristics Clinical Complex Crystallization DNA DNA Footprint DNA Polymerase II DNA mapping DNA-Directed RNA Polymerase Development Disease Electroporation Enzymes Escherichia coli Event Excision Gene Expression Gene Expression Regulation Genes Genetic Transcription Grain Growth and Development function Health High-Throughput Nucleotide Sequencing Human In Vitro Kinetics Knock-out Label Libraries Life Magnetism Modeling Molecular Molecular Biology Techniques Molecular Conformation Molecular Structure Monitor Mutate Organism Osmoregulation Plasmids Process Public Health RNA Regulation Research Personnel Resistance Resolution Role Specificity Structure Techniques Testing Transcript Transcription Initiation Transcriptional Regulation Validation Work antimicrobial base biological adaptation to stress cleavage factor experimental study in vivo insight interest microbial molecular modeling novel novel therapeutic intervention promoter public health relevance simulation single molecule single-molecule FRET stem

项目摘要

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.

项目概要：细菌转录起始和启动子逃逸是基因表达的高度调控的早期步骤。一当新生RNA的5 ′末端与启动子的3.2区发生冲突时，发生关键的起始步骤特异性因子σ70（σR3.2）阻塞RNA出口通道。然后，要么清除阻塞通道，促进RNA通过RNA出口通道和RNAP的正向易位以逃离启动子，或新生的RNA反向易位到NTP进入通道中，导致其流产释放。我们最近表明一小部分RNAP在长时间暂停的回溯中间体中得到稳定，初始化。我们还表明，即使在通过新生RNA从RNA出口通道中去除σR3.2后，转录，转录动力学仍然比预期的延长慢。因此，我们假设另外的启动子逃逸中间体进一步减慢从起始到延伸的转变，并且两种中间体都具有调节作用。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导出的分子内距离作为空间约束，粗粒度的模拟。在目标1.B中，我们将定义控制丰度的分子决定因素这些晚期起始中间体。具体来说，我们将研究σ70区域的序列和顺序，在不同启动子的启动子逃逸过程中从RNA出口通道中除去。在目标1.B中，假设：（1）启动子逃逸过程中σR3和σR4的置换遵循两步过程;（2）在转录泡的挤压DNA模板链中形成的凸起有助于去除这些σ 来自RNA的区域通过突出到通道中而离开通道。我们最近发现，许多RNAP停滞在体内许多基因的启动子上，这些基因对应激反应至关重要，在高渗条件下，Escherichia coli A/Escherichia coli B的失速增强。大肠杆菌菌株（未发表）。在目标2中，我们将测试启动暂停是否发生在活细菌中，并作为调节中间体 for stress压力response反应.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。体内足迹技术。我们还将开发体内smFRET转录气泡大小测定来测试在细菌细胞中是否通过类似于目标1中研究的机制发生起始暂停。该项目将显著推进转录领域，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研究细菌的生物学行为至关重要。有效的抗菌治疗的发展;（2）RNAP结构的许多特征的保护和从细菌到人类的功能促进了真核酶转录机制的建模;（3）暂停-回溯RNAP在起始时的结构尚未确定。因此，阻断不同启动子的RNA出口通道的σ70区域的空间重排将提供有价值的深入了解启动子逃逸的机制。