Burgstaller P, Hermann T, Huber C, Westhof E, Famulok M
Institut für Biochemie der LMU München-Genzentrum, Würmtalstrasse 221, 81375 München, Germany.
Nucleic Acids Res. 1997 Oct 15;25(20):4018-27. doi: 10.1093/nar/25.20.4018.
We have recently shown that isoalloxazine derivatives are able to photocleave RNA specifically at G.U base pairs embedded within a helical stack. The reaction involves the selective molecular recognition of G.U base pairs by the isoalloxazine ring and the removal of one nucleoside downstream of the uracil residue. Divalent metal ions are absolutely required for cleavage. Here we extend our studies to complex natural RNA molecules with known secondary and tertiary structures, such as tRNAs and a group I intron (td). G.U pairs were cleaved in accordance with the phylogenetically and experimentally derived secondary and tertiary structures. Tandem G.U pairs or certain G.U pairs located at a helix extremity were not affected. These new cleavage data, together with the RNA crystal structure, allowed us to perform molecular dynamics simulations to provide a structural basis for the observed specificity. We present a stable structural model for the ternary complex of the G. U-containing helical stack, the isoalloxazine molecule and a metal ion. This model provides significant new insight into several aspects of the cleavage phenomenon, mechanism and specificity for G. U pairs. Our study shows that in large natural RNAs a secondary structure motif made of an unusual base pair can be recognized and cleaved with high specificity by a low molecular weight molecule. This photocleavage reaction thus opens up the possibility of probing the accessibility of G.U base pairs, which are endowed with specific structural and functional roles in numerous structured and catalytic RNAs and interactions of RNA with proteins, in folded RNAs.
我们最近发现，异咯嗪衍生物能够在螺旋堆积中嵌入的G·U碱基对处特异性地光裂解RNA。该反应涉及异咯嗪环对G·U碱基对的选择性分子识别以及尿嘧啶残基下游一个核苷的去除。裂解绝对需要二价金属离子。在此，我们将研究扩展到具有已知二级和三级结构的复杂天然RNA分子，如tRNA和I组内含子（td）。G·U碱基对的裂解符合系统发育和实验推导的二级和三级结构。串联的G·U碱基对或位于螺旋末端的某些G·U碱基对不受影响。这些新的裂解数据，连同RNA晶体结构，使我们能够进行分子动力学模拟，为观察到的特异性提供结构基础。我们提出了一个包含G·U的螺旋堆积、异咯嗪分子和金属离子的三元复合物的稳定结构模型。该模型为G·U碱基对的裂解现象、机制和特异性的几个方面提供了重要的新见解。我们的研究表明，在大型天然RNA中，由不寻常碱基对构成的二级结构基序可以被低分子量分子以高特异性识别和裂解。因此，这种光裂解反应为探测G·U碱基对的可及性开辟了可能性，G·U碱基对在许多结构化和催化性RNA以及RNA与蛋白质的相互作用中具有特定的结构和功能作用，存在于折叠的RNA中。
