An axial binding site in the Tetrahymena precursor RNA.
An axial binding site in the Tetrahymena precursor RNA.
复制标题
四膜虫前体 RNA 中的轴向结合位点。
DOI：
10.1016/0022-2836(91)90590-3
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发表时间：
1991
影响因子：
5.6
通讯作者：
Christian,E
中科院分区：
文献类型：
--
作者：
Yarus,M;Illangesekare,M;Christian,E
先前的研究允许在四膜虫自剪接前核糖体前体RNA的活性位点内构建G和精氨酸结合的三种不同模型。这些模型（碱基三重、轴向 I 和轴向 II）现在通过测量跨位点位置具有核苷酸取代的 RNA 的特异性来区分。因为半保守的未配对核苷酸263对四膜虫RNA的底物或抑制剂选择没有影响，所以我们得出结论，轴向I模型是不可能的。相反，取代的RNA和核苷类似物的数据表明核苷酸265与底物形成氢键。因此，活性位点呈轴向，因为 P7 螺旋 5' 侧的多个核苷酸处存在底物接触。当供体或受体位于RNA上时，无论265号核苷酸是嘌呤还是嘧啶，或者265号核苷酸是错配、摆动配对还是正常配对，都可以观察到该氢键的影响。该模式与轴II模型一致。分子动力学和能量最小化计算得出与这些定点取代相同的结论；基本三重模型和轴向 I 模型动态不稳定。在热搅拌下，第三个模型位置（轴向 II）转变为相关但更稳定的结构，轴向 III。轴向III活性位点的特征在于保守的凸出碱基263从P7螺旋中挤出，通过核苷酸262堆叠形成的G碱基半袋，以及最初为碱基三重位点和轴向II位点提出的与G碱基的所有键的形成。由于这些氢键，轴向 III 位点也与酶特异性数据一致。轴向 III 模型表明 RNA 螺旋凹槽内具有不可预见的口袋形成能力，表明该位点可能异常灵活，并与有关遗传密码起源的假设有关。
Previous studies allow the construction of three distinct models of the binding of G and arginine within the active site of theTetrahymenaself-splicing preribosomal precursor RNA. These models (base triple, axial I and axial II) are now distinguished by measurements on the specificity of RNAs with nucleotide substitutions at positions spanning the site. Because the semi-conserved unpaired nucleotide 263 has no effect on substrate or inhibitor selection by theTetrahymenaRNA we conclude that the axial I model is improbable. In contrast, data with substituted RNAs and nucleoside analogs suggest that nucleotide 265 makes a hydrogen bond with the substrate. Accordingly the active site appears axial because substrate contacts exist at more than one nucleotide on the 5′ side of the P7 helix. The effects of this hydrogen bond are observable in cases where the donor or acceptor is on the RNA, whether nucleotide 265 is a purine or pyrimidine, or whether nucleotide 265 is mispaired, wobble paired or normally paired. This pattern is consistent with the axial II model. Molecular dynamics and energy minimization calculations lead to the same conclusions as these site-directed substitutions; the base triple and axial I models are unstable dynamically. Under thermal agitation, the third model site (axial II) is transformed to a related, but more stable structure, axial III. The axial III active site is characterized by the extrusion of the conserved bulged base 263 from the P7 helix, a semipocket for G base formed by stacking of nucleotide 262, and formation of all bonds to the G base originally proposed for both the base triple and axial II sites. Because of these hydrogen bonds the axial III site is also consistent with data on enzymatic specificity. The axial III model indicates an unforeseen capacity for pocket formation within the groove of an RNA helix, suggests that the site may be unusually flexible, and bears on a hypothesis concerning the origin of the genetic code.