Conformation of the Group II intron branch site in solution.
Conformation of the Group II intron branch site in solution.
复制标题
溶液中 II 组内含子分支位点的构象。
DOI：
10.1021/ja0578754
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发表时间：
2006
影响因子：
15
通讯作者：
Greenbaum,NancyL
中科院分区：
文献类型：
--
作者：
Schlatterer,JorgC;Crayton,SamuelH;Greenbaum,NancyL
II组内含子是多结构域核酶，催化其自身从前体mRNA去除。用于第一切割步骤的亲核试剂是结构域6（D 6）内的特定腺苷的2 'OH，称为分支位点。与真核剪接体的机制相似性和有限的二级结构相似性导致许多人推测这两个系统有共同的祖先。我们通过核磁共振和荧光光谱阐明了ai 5 γ II组内含子D 6内分支位点区域的结构特征和内环对分支位点构象的重要性。其中2-氨基嘌呤取代分支位点腺苷的荧光实验表明，分支位点碱基暴露于溶剂，并且该位置被Mg 2+或Ca 2+增强。位于腺苷分支位点两侧的两个尿苷残基的亚氨基质子的高场NMR化学位移，以及它们之间的ann→n+ 2 NOE，表明两个尿苷具有堆叠的螺旋内构象。相反，缺失内环的突变D 6的NMR和2-氨基嘌呤荧光光谱的结果表明分支位点腺苷的暴露较少的位置，其可能与相对的3 'G形成G-A碱基对。这些研究结果描述了一个模型，其中的分支网站腺苷的D 6是在一个extrahelical位置，由两个intrahelical基地包围。内环和二价金属离子促进了这种基序。
Group II introns are multidomain ribozymes that catalyze their own removal from pre-mRNA. The nucleophile for the first cleavage step is the 2‘OH of a specific adenosine within domain 6 (D6), called the branch site. Mechanistic parallels and limited secondary structural similarity with the eukaryotic spliceosome lead many to speculate that the two systems have a common ancestry. We have elucidated structural features of the branch site region and the importance of the internal loop to branch site conformation within D6 of the ai5γ Group II intron by NMR and fluorescence spectroscopy. Fluorescence experiments in which 2-aminopurine was substituted for the branch site adenosine suggest that the branch site base is exposed to solvent and that this position is enhanced by Mg2+or Ca2+. Upfield NMR chemical shifts of imino protons of the two uridine residues flanking the branch site adenosine, and ann→n+ 2 NOE between them, suggest a stacked intrahelical conformation of the two uridines. In contrast, results of NMR and 2-aminopurine fluorescence spectra of a mutated D6 from which the internal loop had been deleted suggest a less exposed position of the branch site adenosine, which is likely to form a G−A base pair with the opposing 3‘G. These findings describe a model in which the branch site adenosine of D6 is in an extrahelical position, surrounded by two intrahelical bases. The internal loop and divalent metal ions facilitate this motif.