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.

项目摘要：细菌转录起始和启动子逃逸是基因表达的高度调节的早期步骤。一个当新生RNA的5'端与启动子的区域3.2发生冲突时，就会发生关键启动步骤特异性因子σ70（σR3.2）遮挡RNA出口通道。然后，要清除封闭的通道通过RNA出口通道和RNAP促进RNA向前易位，以逃脱启动子或新生的RNA反向转移到NTP入口通道中，导致其流产释放。我们最近有表明，在长期暂停的回溯中间人中，一小部分RNAP被稳定引发。我们还表明，即使在从RNA出口通道中删除σR3.2之后转录本，转录动力学仍然比伸长率预期的要慢。因此，我们假设其他启动子逃生中级进一步减慢了从启动到伸长的过渡，并且两个中间体都有调节作用。在AIM 1.a中，我们将阐明转录启动的结构通过使用多个实验衍生的分子内距离作为空间约束，这些状态中的复合物在粗粒模拟上。在AIM 1.B中，我们将定义控制抽象的分子确定词在这些晚期倡议中间人中。具体而言，我们将检查σ70区域的序列和顺序在启动子逃生期间，从RNA出口通道中删除不同启动子的启动器。在目标1.b中假设：（1）启动子逃脱过程中σR3和σR4的位移遵循了两步的过程；（2）在转录气泡的皱纹DNA模板链中形成的凸起有助于去除这些σ 通过投射到通道中，来自RNA出口通道的区域。我们最近发现过多的体内许多基因启动子的RNAP摊位数对于应力反应至关重要在Δgrea/∆greb大肠杆菌菌株（未发表）的高渗条件下，这种失速得到了增强。在AIM 2中，我们将测试是否暂停在活细菌中发生，并用作调节性中间体应力反应。我们将通过高分辨率（1-2 NT）染色体DNA映射来检验这一假设。体内技术足迹。我们还将开发体内SMFRET转录气泡尺寸测定进行测试是否通过与AIM 1相似的机制在细菌细胞中进行暂停。由于以下原因，该项目将大大提高转录领域：（1）抗生素抗性是一个严重的公共卫生问题。阐明细菌基因调节的机制对于开发有效的抗菌治疗；（2）RNAP结构的许多特征的保存和从细菌到人类的功能促进了真核酶转录机制的建模。（3）尚未确定暂停的RNAP的结构尚未确定。因此，描述 σ70区域阻止不同启动子的RNA出口通道的空间重排将提供有价值的深入了解启动子逃脱的机制。