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Invisible States and Double Conformational Capture in RNA-RNA Recognition: The Docking Transition of the Hairpin Ribozyme

RNA-RNA 识别中的不可见状态和双重构象捕获：发夹核酶的对接转变

基本信息

批准号：
1413356
负责人：
Charles Hoogstraten
金额：
$ 73.57万
依托单位：
Michigan State University
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2019-02-28
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1413356&HistoricalAwards=false
关键词：
Invisible States Double Conformational Capture

项目摘要

Ribonucleic acid (RNA) is most familiar as the messenger that carries genetic information stored in DNA to the sites where it is translated into the proteins that do the work of the cell. In recent years, however, scientists have realized that RNA molecules that do not encode proteins play a wide variety of roles in biology, and that many of these "noncoding RNAs" take up complex and intricate three-dimensional shapes in order to fill their biological function. In this project, the process by which the well-studied RNA enzyme known as the hairpin ribozyme forms its folded shape will be examined in detail. The results will uncover principles for the processes and driving forces of RNA folding that will be of general applicability to other noncoding RNA molecules and will thus enhance scientific understanding of a variety of fundamental cellular processes. In addition, a standard set of baseline technical parameters (NMR frequencies for atoms in RNA molecules that lack three-dimensional structure) will be generated that will be of use to many workers in the field of RNA biophysics. The lab in which this research will be performed has a strong history in the graduate education of underrepresented groups, and this project will continue and extend these efforts to the undergraduate level via coordination with existing recruitment and retention efforts at Michigan State.Although the factors underlying secondary structure (helix formation) in DNA and RNA are relatively well understood, the detailed study of the driving forces and mechanisms of the formation of complex tertiary structures in nucleic acids has not kept pace with the great recent advances in RNA structural biology. The active conformation of the hairpin ribozyme is generated by an interaction between two RNA internal loops to generate an intricate docked structure with significant conformational rearrangements. It is hypothesized that this transition is an example of double conformational capture, in which both loops undergo fluctuations to conformation(s) resembling their docked forms and only collisions between loops that are each sampling those forms are productive for recognition. This hypothesis will be tested using NMR spin-relaxation studies of each loop in isolation. Such experiments can identify minor conformers, or "invisible states," sampled by each loop, and the hypothesis predicts that the chemical shifts of these invisible states will resemble those of the docked form. Recently- reported advanced chemical shift calculation methods will be used to estimate the chemical shift changes expected upon transition to a docked-like state. To rule out simple molecular unfolding, a systematic determination of the NMR chemical shifts characteristic of the unfolded state of RNA will be performed, thus producing a reference database likely to be broadly useful to workers in the field. Observed chemical shift changes to the invisible state(s) will be compared to those expected from both fluctuations to the docked-like conformation (as hypothesized) and to simple unfolded states. Finally, advanced molecular mechanics calculations will be used to map out the details of the free-energy landscape implied by the NMR experiments and to verify the existence of energetically accessible trajectories corresponding to the proposed pathways.

核糖核酸（RNA）作为信使最为人所熟知，它将储存在DNA中的遗传信息携带到被翻译成细胞中工作的蛋白质的位置。然而，近年来，科学家们已经意识到，不编码蛋白质的RNA分子在生物学中发挥着各种各样的作用，并且许多这些“非编码RNA”采取复杂和复杂的三维形状，以填补其生物学功能。在这个项目中，被称为发夹核酶的RNA酶形成折叠形状的过程将被详细研究。这些结果将揭示RNA折叠过程和驱动力的原理，这些原理将普遍适用于其他非编码RNA分子，从而增强对各种基本细胞过程的科学理解。此外，将生成一套标准的基线技术参数（缺乏三维结构的RNA分子中原子的核磁共振频率），这将对RNA生物物理学领域的许多工作人员有用。进行这项研究的实验室在弱势群体的研究生教育方面有着悠久的历史，该项目将通过与密歇根州立大学现有的招聘和保留努力的协调，继续并将这些努力扩展到本科生水平。尽管人们对DNA和RNA的二级结构（螺旋结构）的潜在因素了解得相对较好，但对核酸中复杂三级结构形成的驱动力和机制的详细研究还没有跟上RNA结构生物学近年来的巨大进展。发夹核酶的活性构象是由两个RNA内部环之间的相互作用产生的，以产生具有显着构象重排的复杂停靠结构。据推测，这种转变是双构象捕获的一个例子，其中两个环都经历类似于其停靠形式的构象的波动，并且只有每个采样这些形式的环之间的碰撞才能产生识别。这一假设将使用核磁共振自旋松弛研究的每个环在隔离中进行测试。这样的实验可以识别小的构象，或“看不见的状态”，由每个回路采样，并且假设预测这些看不见的状态的化学变化将类似于那些停靠形式。最近报道的先进的化学位移计算方法将用于估计化学位移的变化，预期过渡到一个停靠的状态。为了排除简单的分子展开，将对RNA展开状态的核磁共振化学位移特征进行系统的测定，从而产生一个可能对该领域的工作人员广泛有用的参考数据库。观察到的到不可见状态的化学位移变化将与预期的从船坞状构象的波动（假设）和简单的未展开状态的变化进行比较。最后，先进的分子力学计算将用于绘制核磁共振实验所隐含的自由能景观的细节，并验证与所提出的路径相对应的能量可达轨迹的存在。