Search for the Structural Basis of Biomacromolecular Function and Activity

在过去的一年里，我们一直专注于两个领域的研究。1.研究REV反应元件(RRE)的结构生物学；2.研究腺嘌呤核糖开关的结构和构象；3.发展一种选择性标记RNA分子的技术。1.HIV-1mRNA的核输出需要病毒蛋白REV与病毒RNA中的REV反应元件(RRE)的特异性相互作用和协同寡聚。RRE的结构已经研究了二十多年，但它的三维结构仍然没有解决。本文报道了一种基于小角X射线散射(SAXS)分析的20埃分辨率的RRE溶液结构。RRE采用了“A”型结构，其中两条腿将两个已知的REV结合位点定位在55埃之间，与REV二聚体中两个RNA结合基序之间的距离相匹配。突变和功能研究表明，RRE的机制和拓扑作用是在最佳距离上定位两个REV结合位点，以实现与REV蛋白的特异性和协同性相互作用。2.核糖开关是一种功能性信使RNA，通过构象变化调节基因表达。此外，核糖开关的三级结构也依赖于阳离子浓度，因为RNA主干是高度负电荷的。X射线溶液在小角(SAXS)和广角(WAXS)中的散射对理解结构、动力学和功能之间的联系产生了越来越大的影响。SAXS数据提供了RNA的大小、重量和形状信息，而WAXS数据对溶液中的微小构象变化很敏感，并提供了一种表征溶液中RNA构象空间的手段。结合计算方法，我们发现小角和广角X射线散射(SAXS和WAXS)可以作为配体和离子浓度的函数来探索腺嘌呤核糖开关适配子结构域的构象。在原子水平上表征了多个折叠态。我们的结果表明，结合口袋改变了它的构象对配体结合的反应。我们的结果表明，腺嘌呤核糖开关适配子的远端环-环相互作用限制了三向连接的构象自由度，促进了配体结合；在生理条件下，配体结合也限制了三向连接的自由度，促进了远端环-环相互作用。这些结果提供了腺嘌呤核糖开关适配子在原子水平上的分级折叠随配体和离子浓度的函数的综合视图。3.本课题组发展了一种利用固相多循环酶促反应选择性标记大分子RNA指定残基(S)和/或片段(S)的新方法。由于RNA在生物学中扮演着广泛的角色，因此SLOR的潜在应用是广泛和深远的。以下只是几个方面的例子。普通RNA生物化学、生物物理学和分子生物学。荧光标记的RNA分子可用于研究RNA之间、RNA与DNA、RNA与蛋白质之间的相互作用，以及显微注射后的细胞环境中的相互作用。例如，选择性标记的RNA可用于研究基因表达调控中的核糖开关机制。使用SLOR可以将荧光残基掺入核糖开关中的两个战略位置，以便利用时间分辨的单分子Forster共振能量转移(FRET)实验监测与适体和表达平台结构域之间的相对运动直接同步的开关事件。由于缺乏特定的荧光标记的核糖开关RNA分子，探测这样的事件是不可能的。RNA结构生物学。仅靠RNA分子几乎不可能结晶以确定结构。核磁共振波谱是确定RNA结构的理想方法，因为它是一种溶液状态的方法，不需要结晶。然而，由于化学位移信号的广泛重叠和核磁共振信号的短寿命，它仅限于小RNA，最多50个残基。有了SLOR，可以使用指定残基(S)和/或片段(S)上的选择性标记RNA来记录核磁共振信号，从而大大简化了核磁共振谱，便于直接解释。此外，人们可以选择性地对指定的残基/片段进行氢化，并记录来自剩余残基的信号。来自剩余残基的信号将具有更长的寿命，从而显著提高核磁共振信号的分辨率和灵敏度。这种增强将使使用核磁共振光谱学确定更大RNA的高分辨率结构成为可能：这将使RNA结构生物学发生革命性变化。SLOR方法将对我的团队和NCI内其他几个团队之间的几个合作产生直接影响(这些合作是我开发SLOR的部分原因)。目前，我们已经为SLOR技术申请了临时专利。

During the past year, we have been focusing on two areas of research. 1. Study structural biology of the Rev response element (RRE); 2. Study the structure and conformation of adenine riboswitch; 3. develop a technology for selective labeling of RNA molecules. 1. Nuclear export of HIV-1 mRNA requires specific interaction and cooperative oligomerization of the viral protein Rev with the Rev response element (RRE) in viral RNA. The structure of the RRE has been studied for more than two decades but its three dimensional structure remains unsolved. Here we report an 20 Angstrom-resolution solution structure of the RRE based on small-angle X-ray scattering (SAXS) analysis. The RRE adopts an "A"-like structure in which the two legs position the two known Rev binding sites 55 Angstrom apart, matching the distance between the two RNA-binding motifs in the Rev dimer. Mutational and functional studies indicate that the mechanistic and topological role of the RRE is to position two tracks of Rev binding sites at an optimal distance for specific and cooperative interaction with the Rev proteins. 2. Riboswitches are functional messenger RNA that regulates gene expression through conformational changes. In addition, riboswitch tertiary structure is also dependent on cation concentration as the RNA backbone is highly negatively charged. X-ray solution scattering in both small-angle (SAXS) and wide-angle (WAXS) is making an increasing impact on the understanding of the linkage between structure, dynamics and function. SAXS data provides RNA size, weight and shape information, and WAXS data are sensitive to small conformation changes in solution and provides a means to characterize the RNA conformational space in solution. We show small-angle and wide-angle X-ray scattering (SAXS &WAXS) can be used to probe Adenine riboswitch aptamer domain conformations as a function of ligand and ion concentration by combining with computational approaches. Multiple folding states were characterized in atomic levels. Our results revealed that the binding pocket changed its conformation in response to ligand binding. Our results suggest that the distal loop-loop interaction of Adenine riboswitch aptamer restricts the conformational freedom of the three-way junction to promote ligand binding; the ligand binding also restricts the freedom of three-way junction to promote the distal loop-loop interaction under physiological conditions. These results provide an integrated view of hierarchical folding in atomic levels in an adenine riboswitch aptamer as a function of ligand and ion concentration. 3. My group has developed a new method for selective labeling of RNA (SLOR) at designated residue(s) and/or segment(s) of large RNAs using solid-phase multi-cycle enzymatic reactions. The potential applications of SLOR are broad and far reaching, due to a wide range of roles that RNA plays in biology. The followings are just examples of a few areas. General RNA biochemistry, biophysics and molecular biology. The fluorescent labeled RNA molecules can be used to study interaction between/within RNAs, and between RNA and DNA, RNA and proteins in vitro or within the cellular environment following microinjection. For example, selectively labeled RNA can be used to study riboswitch mechanisms in regulation of gene expression. The fluorescent residues can be incorporated at two strategically locations in the riboswitch using SLOR in order to monitor the switching event that is directly synchronized with the relative movement between the aptamer and expression platform domains using time-resolved single molecule Forster Resonance Energy Transfer (FRET) experiments. Probing such an event has not been possible because of lack of the specifically fluorescent labeled riboswitch RNA molecules. RNA structural biology. RNA molecules alone are almost impossible to crystallize for structure determination. NMR spectroscopy is an ideal method for structure determination of RNAs since it is a solution-state method and does not require crystallization. However, it is limited to only small RNAs, up to 50 residues, because of the extensive overlaps of chemical shift signals and short lifetimes of NMR signals. With SLOR, selectively labeled RNAs at designated residue(s) and/or segment(s) can be used for recording NMR signals, resulting in greatly simplified NMR spectra for straightforward interpretation. Moreover, one can selectively deuterate designated residues/segments and record the signals from the remaining residues. The signals from the remaining residues will have a much longer lifetime, resulting in significant enhancement of both resolution and sensitivity of NMR signals. This enhancement will make it possible to determine high-resolution structures of much larger RNAs using NMR spectroscopy: this will revolutionize RNA structural biology. The SLOR method will have an immediate impact to several collaborations between my group and several other groups within NCI (those collaborations are part of reasons that I developed SLOR). Currently, we have filed a provisional patent for the SLOR technology.