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，新生的RNA二级结构可以对转录产生重大影响，例如作为内在终止子的关键组成部分的发夹。此外，在转录过程中发生的按时间顺序的、定向的RNA合成经常产生RNA折叠，而不是全长转录本的热力学最稳定的结构。转录和功能RNA折叠之间的这种耦合只是一个新兴领域的一个例子，该领域试图了解基因表达和RNA结构之间的关系。在这种关系的一个很好的例子中，细菌核糖开关含有非编码RNA“适配子”，其二级和三级结构随着小分子代谢物的结合而重新折叠，通过对转录终止或翻译启动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，使它们成为具有潜在现实世界影响的药物靶点的优秀模型系统。到目前为止，对核糖开关的研究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研究，以及利用核糖开关结合到报告结构中进行基因调控的体内研究。虽然这使得人们对适配子感知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，但对于它们调节的大分子复合体中的核糖开关行为仍然知之甚少。我们将通过研究一种有利的小核糖开关来填补这一空白，该开关可以调节7-氨甲基-7-去氮鸟嘌呤(PreQ1)结合反应中转录终止的效率。为此，我们将利用生物物理和生化工具的独特组合来研究活性转录复合体中的核糖开关。我们将对暂停的转录复合体进行单分子荧光共振能量转移(SmFRET)测量，该复合体由DNA气泡和与RNA聚合酶结合的荧光团标记的新生核糖开关转录本组成，确定下游RNA序列和聚合酶对适体结构和动力学的影响(特定目标1)。我们将使用最近开发的一种称为RNA瞬时结构的单分子动力学分析(SIM-KARTS)的技术来探索在预合成和主动转录的RNA表达平台上终止子和反终止子发夹的相对形成，确定共转录折叠在核糖开关功能中的作用(特定目标2)。最后，我们将结合体外转录实验和smFRET来研究转录因子NusA和RfaH的终止效应，这两个转录因子已经被证明影响新生RNA结构的形成(特定目标3)。除了促进我们对核糖开关的理解外，这些研究还有可能改变我们对RNA结构形成的一般理解，以及RNA结构如何与大分子机器的功能耦合的理解。

 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.