Davis Jared H, Tonelli Marco, Scott Lincoln G, Jaeger Luc, Williamson James R, Butcher Samuel E
Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr. Madison, WI 53706, USA.
J Mol Biol. 2005 Aug 12;351(2):371-82. doi: 10.1016/j.jmb.2005.05.069.
Tertiary interactions are critical for proper RNA folding and ribozyme catalysis. RNA tertiary structure is often condensed through long-range helical packing interactions mediated by loop-receptor motifs. RNA structures displaying helical packing by loop-receptor interactions have been solved by X-ray crystallography, but not by NMR. Here, we report the NMR structure of a 30 kDa GAAA tetraloop-receptor RNA complex. In order to stabilize the complex, we used a modular design in which the RNA was engineered to form a homodimer, with each subunit containing a GAAA tetraloop phased one helical turn apart from its cognate 11-nucleotide receptor domain. The structure determination utilized specific isotopic labeling patterns (2H, 13C and 15N) and refinement against residual dipolar couplings. We observe a unique and highly unusual chemical shift pattern for an adenosine platform interaction that reveals a spectroscopic fingerprint for this motif. The structure of the GAAA tetraloop-receptor interaction is well defined solely from experimental NMR data, shows minor deviations from previously solved crystal structures, and verifies the previously inferred hydrogen bonding patterns within this motif. This work demonstrates the feasibility of using engineered homodimers as modular systems for the determination of RNA tertiary interactions by NMR.
三级相互作用对于RNA的正确折叠和核酶催化至关重要。RNA三级结构通常通过由环-受体基序介导的长程螺旋堆积相互作用而凝聚。通过环-受体相互作用展示螺旋堆积的RNA结构已通过X射线晶体学解析，但尚未通过核磁共振（NMR）解析。在此，我们报道了一个30 kDa的GAAA四环-受体RNA复合物的NMR结构。为了稳定该复合物，我们采用了一种模块化设计，其中RNA被设计成形成同二聚体，每个亚基包含一个GAAA四环，与它的同源11核苷酸受体结构域相隔一个螺旋圈。结构测定利用了特定的同位素标记模式（2H、13C和15N）并针对剩余偶极耦合进行了精修。我们观察到一种独特且非常不寻常的腺苷平台相互作用的化学位移模式，揭示了该基序的光谱指纹。GAAA四环-受体相互作用的结构仅从实验NMR数据就得到了很好的定义，与先前解析的晶体结构有微小偏差，并验证了该基序内先前推断的氢键模式。这项工作证明了使用工程化同二聚体作为模块化系统通过NMR测定RNA三级相互作用的可行性。
