Tuschl T, Gohlke C, Jovin T M, Westhof E, Eckstein F
Max-Planck-Institut für Experimentelle Medizin, Göttingen, Germany.
Science. 1994 Nov 4;266(5186):785-9. doi: 10.1126/science.7973630.
For the understanding of the catalytic function of the RNA hammerhead ribozyme, a three-dimensional model is essential but neither a crystal nor a solution structure has been available. Fluorescence resonance energy transfer (FRET) was used to study the structure of the ribozyme in solution in order to establish the relative spatial orientation of the three constituent Watson-Crick base-paired helical segments. Synthetic constructs were labeled with the fluorescence donor (5-carboxyfluorescein) and acceptor (5-carboxytetramethylrhodamine) located at the ends of the strands constituting the ribozyme molecule. The acceptor helix in helix pairs I and III and in II and III was varied in length from 5 to 11 and 5 to 9 base pairs, respectively, and the FRET efficiencies were determined and correlated with a reference set of labeled RNA duplexes. The FRET efficiencies were predicted on the basis of vector algebra analysis, as a function of the relative helical orientations in the ribozyme constructs, and compared with experimental values. The data were consistent with a Y-shaped arrangement of the ribozyme with helices I and II in close proximity and helix III pointing away. These orientational constraints were used for molecular modeling of a three-dimensional structure of the complete ribozyme.
为了理解RNA锤头状核酶的催化功能，三维模型至关重要，但目前尚无晶体结构或溶液结构。荧光共振能量转移（FRET）被用于研究溶液中核酶的结构，以确定三个组成的沃森-克里克碱基配对螺旋片段的相对空间取向。合成构建体用位于构成核酶分子的链末端的荧光供体（5-羧基荧光素）和受体（5-羧基四甲基罗丹明）进行标记。螺旋对I和III以及II和III中的受体螺旋长度分别从5到11个碱基对和5到9个碱基对不等，并测定了FRET效率，并与一组标记的RNA双链体参考数据相关联。基于向量代数分析，根据核酶构建体中相对螺旋取向预测FRET效率，并与实验值进行比较。数据与核酶的Y形排列一致，其中螺旋I和II紧密相邻，螺旋III指向外侧。这些取向限制被用于完整核酶三维结构的分子建模。
