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

核糖核酸（RNA）是最熟悉的信使，它携带储存在DNA中的遗传信息，将其翻译成蛋白质，完成细胞的工作。然而，近年来，科学家们已经意识到，不编码蛋白质的RNA分子在生物学中扮演着各种各样的角色，并且这些“非编码RNA”中的许多都占据了复杂而错综复杂的三维形状，以填补其生物学功能。在这个项目中，将详细研究被称为发夹状核酶的RNA酶形成折叠形状的过程。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，将产生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NMR频率），这将对RNA生物物理学领域的许多工作者有用。进行这项研究的实验室在代表性不足的群体的研究生教育方面有着悠久的历史，本项目将通过与密歇根州立大学现有的招聘和保留工作协调，继续并将这些努力扩展到本科阶段。DNA和RNA中的（螺旋形成）是相对较好理解的，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最近的重大进展。RNA结构生物学进展发夹核酶的活性构象是通过两个RNA内环之间的相互作用产生的，以产生具有显著构象重排的复杂对接结构。假设这种转变是双重构象捕获的一个例子，其中两个环都经历类似于其对接形式的构象的波动，并且只有每个都对这些形式进行采样的环之间的碰撞才能产生识别。这一假设将测试使用NMR自旋弛豫研究的每一个孤立的循环。这样的实验可以识别次要的构象，或“不可见的状态”，通过每个循环采样，该假设预测这些不可见状态的化学位移将类似于对接形式的化学位移。最近报道的先进化学位移计算方法将用于估计在过渡到对接样状态时预期的化学位移变化。为了排除简单的分子解折叠，将进行RNA解折叠状态的NMR化学位移特性的系统测定，从而产生可能对该领域的工作者广泛有用的参考数据库。观察到的化学位移变化的不可见的状态（S）将进行比较，从两个波动的对接样构象（假设）和简单的未折叠状态的预期。最后，先进的分子力学计算将被用来绘制出NMR实验所暗示的自由能景观的细节，并验证存在的能量可访问的轨迹对应于拟议的途径。

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.