Cotranscriptional folding of single riboswitches

 描述（由申请人提供）：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RNA二级结构可以对转录具有显著影响，如作为内在终止子的关键组分的发夹所例示。此外，在转录过程中发生的时间有序的定向RNA合成通常产生RNA折叠，而不是全长转录物的最稳定结构。转录和功能性RNA折叠之间的这种耦合仅仅是一个新兴领域的一个例子，该领域试图理解基因表达和RNA结构之间的关系。在这种关系的一个优雅的例子中，细菌核糖开关含有非编码RNA“适体”，其二级和三级结构响应于小代谢物的结合而重新折叠，通过对转录终止或翻译起始的影响导致下游基因表达的变化。核糖开关是细菌基因调控的关键机制，在某些物种中，它们负责调节高达4%的所有基因，使其成为具有作为药物靶标的现实影响潜力的优秀模型系统。核糖开关的研究迄今已被分为两个独立的调查领域：分离的适体结构域的结构和生物物理学研究，并在体内研究的基因调控核糖开关纳入报告构建体。虽然这导致了广泛的知识的机制，适体感测它们的配体和许多新的调节RNA序列的发现，宝贵的核糖开关行为的背景下，他们调节的大分子复合物仍然知之甚少。我们将填补这一空白，通过研究一个有利的小核糖开关，调节转录终止的效率，响应7-氨甲基-7-脱氮鸟嘌呤（preQ 1）的结合。为此，我们将利用生物物理和生物化学工具的独特组合来研究活性转录复合物中的核糖开关。我们将进行单分子荧光共振能量转移（smFRET）测量暂停转录复合物组成的DNA气泡和荧光标记的新生核糖开关转录结合RNA聚合酶，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）。我们将使用我们最近开发的称为RNA瞬时结构的单分子动力学分析（SiM-KARTS）的技术来探测在预合成以及活跃转录的RNA的表达平台中终止子和抗终止子发夹的相对形成，确定核糖开关功能中共转录折叠的作用（特异性目的2）。最后，我们将结合联合收割机体外转录试验和smFRET研究转录因子NusA和RfaH的终止效应，这两种转录因子已被证明会影响新生RNA结构的形成（特异性目标3）。除了推进我们对核糖开关的理解，这些研究有可能改变我们对RNA结构形成的理解，以及RNA结构如何与大分子机器的功能相结合。

 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.