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（由适用提供）：该提案的最终目标是解开RNA转录和折叠之间的耦合。众所周知，新生的RNA二级结构可以对转录产生重大影响，这是由发夹作为内在终结剂的关键组成部分的例证。此外，在转录过程中发生的时间订购的定向RNA合成通常会产生除全长转录本最热力学稳定结构以外的RNA折叠。转录和功能RNA折叠之间的这种耦合只是新兴领域中试图了解基因表达与RNA结构之间关系的一个例子。在这种关系的优雅例子中，细菌核糖开关包含非编码RNA“适体”，其二级和三级结构响应于小型代谢物的结合而重折，从而导致下游基因表达通过对转录终止或翻译启动的影响而变化。核糖开关是细菌基因调节的关键机制，在某些物种中，它们负责调节所有基因的4％，使它们具有出色的模型系统，具有现实世界中影响作为药物靶标的潜力。到目前为止，对核糖开关的研究已经分为两个独立的询问领域：分离的apatamar结构域的结构和生物物理研究，以及使用纳入报道构建体中的核糖开关进行基因调节的体内研究。尽管这导致了人们对适体感觉配体和发现许多新调节RNA序列的机制的广泛了解，但在它们调节的大分子复合物的背景下，珍贵的核糖开关行为仍然很少知道。我们将通过研究一个有利的核糖开关来填补这一空白，该核糖开关调节转录终止的效率，以响应7-氨基甲基-7-二唑宁（PREQ1）结合。为此，我们将利用生物物理和生化工具的独特组合来研究主动转录复合物中的核糖开关。我们将对由DNA气泡和与RNA聚合酶结合的荧光团新生的核糖开关转录本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，确定下游RNA序列和聚合酶对ATAMER结构和动力学的影响（特定特定目标1）。我们将使用最近开发的RNA瞬态结构（SIM-KARTS）的称为单分子动力学分析的技术来探测终结剂和抗固定器发夹的相对形成，并在同步预先转录的RNA的表达平台中，并确定Co-Transscriptscript折叠的作用，以确定Co-Transsscriptional折叠的作用，具体折叠的作用（具体核能WitchItch inter（特定于Aim 2）。最后，我们将结合体外转录分析和SMFRET，以研究转录因子NUSA和RFAH的终止效应，这些终止效应已证明会影响新生的RNA结构形成（特定目标3）。除了促进我们对核糖开关的理解外，这些研究还可以改变我们对RNA结构形成的理解，以及如何将RNA结构与大分子机器的功能耦合。