Fountain M A, Serra M J, Krugh T R, Turner D H
Department of Chemistry, University of Rochester, New York 14627-0216, USA.
Biochemistry. 1996 May 28;35(21):6539-48. doi: 10.1021/bi952697k.
The hairpin loop GUAAUA occurs frequently in ribosomal RNA. Optical melting studies show that r(GGCGUAAUAGCC) folds into a hairpin containing this loop. The structural features of the r(GGCGUAAUAGCC) hairpin have been determined by NMR and molecular modeling. NOEs from G4-H1' to A9-H2 and from A9-H2 to G10-H1' show that G4 and A9 form a sheared base pair with two hydrogen bonds: A-N7 to G-NH2 and A-NH6 to G-N3. One-dimensional NOE data show no NOEs between the imino protons of U5 and U8, but NOEs are observed between the U5-H1' and the U8-H6 and U8-H5, thus orienting the U8 imino proton away from U5. Thus U5 and U8 do not form an imino hydrogen-bonded U.U pair. The U5-H2' exhibits NOEs to both the A6-H8 and A7-H8, and the 3' phosphorus resonances of U5 and A6 are shifted downfield. This suggests that the helix turn is between the U5 and A6 nucleotides. The JH1'-H2 and JH3'-H4' coupling constants indicate that the loop is dynamic, particularly at 35 degrees C, well below the melting temperature of 63 degrees C. Structures were generated using 75 distance and 46 dihedral angle restraints. In these structures, the U5 base is stacked on the sheared base pair formed by G4 and A9 and can initiate a uridine turn similar to that observed in the anticodon loop of tRNA. The A6, A7, and U8 bases can stack on one another with their hydrogen-bonding surfaces exposed to the solvent, suggesting that they are available for tertiary interactions or protein recognition in rRNA. A range of loop structures are consistent with the data, however. The lack of formation of a U.U mismatch is consistent with a recent model that predicts the stability of hairpin loops of six nucleotides on the basis of the closing base pair and first mismatch in the loop [Serra, M. J., Axenson, T. J., & Turner, D. H. (1994) Biochemistry 33, 14289-14296].
发夹环GUAAUA在核糖体RNA中频繁出现。光学熔解研究表明，r(GGCGUAAUAGCC)折叠成一个包含该环的发夹结构。r(GGCGUAAUAGCC)发夹的结构特征已通过核磁共振（NMR）和分子建模确定。从G4-H1'到A9-H2以及从A9-H2到G10-H1'的核Overhauser效应（NOE）表明，G4和A9形成了一个具有两个氢键的剪切碱基对：A的N7与G的NH2以及A的NH6与G的N3。一维NOE数据显示U5和U8的亚氨基质子之间没有NOE，但在U5-H1'与U8-H6和U8-H5之间观察到了NOE，从而使U8的亚氨基质子远离U5。因此，U5和U8没有形成亚氨基氢键连接的U.U对。U5-H2'与A6-H8和A7-H8都表现出NOE，并且U5和A6的3'磷共振向低场移动。这表明螺旋转折位于U5和A6核苷酸之间。JH1'-H2和JH3'-H4'耦合常数表明该环是动态的，特别是在35摄氏度时，远低于63摄氏度的熔解温度。使用75个距离和46个二面角约束生成了结构。在这些结构中，U5碱基堆积在由G4和A9形成的剪切碱基对上，并且可以引发类似于在tRNA反密码子环中观察到的尿苷转折。A6、A7和U8碱基可以彼此堆积，它们的氢键表面暴露于溶剂中，这表明它们可用于rRNA中的三级相互作用或蛋白质识别。然而，一系列环结构与数据是一致的。未形成U.U错配与最近的一个模型一致，该模型根据环中的封闭碱基对和第一个错配预测六个核苷酸的发夹环的稳定性[Serra, M. J., Axenson, T. J., & Turner, D. H. (1994) Biochemistry 33, 14289 - 14296]。
