Cotranscriptional folding of single riboswitches

单个核糖开关的共转录折叠

基本信息

批准号：
9357619
负责人：
NILS G WALTER
金额：
$ 30.37万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-23 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9357619
关键词：
5&apos Untranslated Regions 7-deazaguanine Affect Anabolism Antibiotics Area Bacillus subtilis Bacteria Bacterial RNA Behavior Binding Biological Biological Assay Biological Models Biological Process Collaborations Complex Computer Simulation Coupled Coupling Crystallography DNA DNA-Directed RNA Polymerase Data Development Drug Targeting Elements Ensure Enzymes Equilibrium Family Fingerprint Fluorescence Fluorescence Microscopy Gene Expression Gene Expression Regulation Genes Genetic Transcription Goals Homologous Gene Human Hybrids Immobilization In Vitro Introns Kinetics Knowledge Label Laboratories Length Letters Ligands Macromolecular Complexes Measurement Measures Messenger RNA Molecular Molecular Conformation Molecular Models Nucleic Acids Nucleoside Q Nucleotides Oligonucleotides Organism Pathway interactions Play Positioning Attribute Proteins RNA RNA Folding RNA Sequences RNA analysis RNA chemical synthesis Reporter Ribonucleic Acid Regulatory Sequences Role Slide Structure Techniques Terminator Regions Testing Thermodynamics Time Transcript Transcription Elongation Transfer RNA Translation Initiation Untranslated RNA Up-Regulation Work antitermination aptamer base biochemical tools biophysical analysis biophysical tools fluorophore in vivo innovation insight molecular modeling nucleobase promoter public health relevance response scaffold single molecule single-molecule FRET success tool transcription factor transcription termination

项目摘要

DESCRIPTION (provided by applicant): The ultimate goal of this proposal is to unravel the coupling between RNA transcription and folding. It is well known that nascent RNA secondary structure can have a significant impact on transcription, as exemplified by the hairpin that acts as a key component of intrinsic terminators. Furthermore, the time-ordered, directional RNA synthesis that occurs during transcription often yields RNA folds other than the most thermodynamically stable structure of the full-length transcript. This coupling between transcription and functional RNA folding is merely one example in an emerging field that seeks to understand the relationship between gene expression and RNA structure. In an elegant example of this relationship, bacterial riboswitches contain non-coding RNA "aptamers" whose secondary and tertiary structures re-fold in response to binding of a small metabolite, leading to a change in expression of the downstream gene through effects on transcription termination or translation initiation. Riboswitches are a key mechanism of gene regulation in bacteria where, in some species, they are responsible for the regulation of up to 4% of all genes, rendering them excellent model systems with potential for real-world impact as drug targets. The study of riboswitches has so far been divided into two separate areas of inquiry: the structural and biophysical studies of isolated aptamer domains, and in vivo studies of gene regulation using riboswitches incorporated into reporter constructs. While this has led to extensive knowledge of the mechanisms by which aptamers sense their ligands and the discovery of many new regulatory RNA sequences, precious little is still known about riboswitch behavior in the context of the macromolecular complexes that they regulate. We will fill this gap through study of a favorably small riboswitch that regulates the efficiency of transcription termination in response t 7-aminomethyl-7-deazaguanine (preQ1) binding. To do so, we will leverage a unique combination of biophysical and biochemical tools to study the riboswitch in active transcription complexes. We will perform single molecule fluorescence resonance energy transfer (smFRET) measurements on paused transcription complexes consisting of a DNA bubble and a fluorophore-labeled nascent riboswitch transcript bound to RNA polymerase, determining the effects of downstream RNA sequence and polymerase on aptamer structure and dynamics (Specific Aim 1). We will use a technique we recently developed termed Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) to probe the relative formation of terminator and antiterminator hairpins in the expression platforms of pre-synthesized as well as actively transcribed RNA, determining the role of co-transcriptional folding in riboswitch function (Specific Aim 2). Finally, we will combine in vitro transcription assays and smFRET to study the termination effects of transcription factors NusA and RfaH, which have been shown to affect nascent RNA structure formation (Specific Aim 3). In addition to advancing our understanding of riboswitches, these studies have the potential to transform our understanding of RNA structure formation in general, and of how RNA structure is coupled to the function of macromolecular machines.

描述（由申请人提供）：该提案的最终目标是阐明 RNA 转录和折叠之间的耦合，众所周知，新生 RNA 二级结构可以对转录产生重大影响，如充当发夹的例子。此外，转录过程中发生的按时间顺序定向的 RNA 合成通常会产生除全长转录物热力学最稳定的结构之外的 RNA 折叠。这种转录与功能性 RNA 之间的耦合。折叠只是寻求了解基因表达与 RNA 结构之间关系的新兴领域的一个例子，细菌核糖开关包含非编码 RNA“适体”，其二级和三级结构会相应地重新折叠。核糖开关是细菌基因调控的关键机制，在某些物种中，它们负责向上的调控。到占所有基因的 4%，使它们成为优秀的模型系统，具有作为药物靶点对现实世界产生影响的潜力。迄今为止，核糖开关的研究已分为两个独立的研究领域：分离适体结构域的结构和生物物理学研究，以及核糖开关的研究。使用整合到报告构建体中的核糖开关进行基因调控的体内研究虽然这使得人们对适体感知其配体的机制有了广泛的了解，并发现了许多新的调节RNA序列，但人们对核糖开关在核酸中的行为仍然知之甚少。我们将通过研究一种可调节 7-氨甲基-7-脱氮鸟嘌呤 (preQ1) 结合的转录终止效率的核糖开关来填补这一空白。结合生物物理和生化工具来研究活性转录复合物中的核糖开关我们将对由 DNA 气泡和组成的暂停转录复合物进行单分子荧光共振能量转移 (smFRET) 测量。荧光团标记的新生核糖开关转录物与 RNA 聚合酶结合，确定下游 RNA 序列和聚合酶对适体结构和动力学的影响（具体目标 1）。 SiM-KARTS）探测预合成和活跃转录 RNA 表达平台中终止子和反终止子发夹的相对形成，确定最后，我们将结合体外转录测定和 smFRET 来研究转录因子 NusA 和 RfaH 的终止效应，这些因子已被证明会影响新生 RNA 结构的形成（具体目标 3）。除了增进我们对核糖开关的理解之外，这些研究还有可能改变我们对 RNA 结构形成以及 RNA 结构如何与大分子功能耦合的理解。机器。