NMR STRUCT OF EUKARYOTIC RNA SELENOCYSTEINE INSERTION ELEMENT (SECIS)

有三类重新编码：核糖体移码， 终止密码子通读和绕过内部mRNA区域。 重新编码 说明书可以包括mRNA内的RNA结构。 这些 结构可以动力学地改变mRNA通过 核糖体，可以直接与核糖体连接， 蛋白质或其他辅助因子，并将它们带到适当的位置， 重新编码事件。 在通用密码中，UGA密码子发出 终止蛋白质合成。 此事件可以在 三种方式，包括硒代半胱氨酸的共翻译插入， 这个三元组（通读的特殊情况）。 对于硒代半胱氨酸 在真核生物中，在终止密码子处插入，有四个要求： (1)合成硒代半胱氨酸所必需的两种酶 氨基酸;（2）一种特殊的tRNA;（3）一种RNA茎环结构，称为 硒代半胱氨酸插入元件（SECIS）是3'-UTR中所必需的 （4）一种蛋白质，它可以作为mRNA的同源物， 原核SelB蛋白（SBP）被认为与SECIS结合， 为硒代半胱氨酸插入核糖体编程。 NMR结构 对这种结构的研究将有助于理解一种元素 在特定mRNA的3'-UTR "通知"核糖体翻译， 与所有UGA密码子相同的mRNA，除了末端 密码子，应指定硒代半胱氨酸插入。 两款车型 SECIS二级结构可用。 两个模型都确定了两个 茎被一内部圈分开，除了一顶端圈。 系统发育鉴定了环II中的三个连续腺苷， 运行或4个碱基对的"核心"区域是必不可少的 SECIS的功能。 茎II的碱基配对寄存器不同， 这两种模型由一个核苷酸组成，包括两个串联的G-A， 建议使用N6-A至N3-G和N7-A至N2-G形成 氢键，为U-U的受限几何结构提供空间 串联体两侧的碱基对 这些有趣的螺旋状 排列是特异性识别SelB的理想位点 同源物（SBP）蛋白用于真核半胱氨酸插入。 A43 已经产生了对应于茎II/环II的核苷酸RNA， NMR研究使用径流T7转录结合双 核酶切割以产生均质RNA。 Simino共振 该RNA的1D 1H光谱位于化学位移范围内 G-A碱基对的预期。 此属性强烈建议模型B 与该SECIS RNA的茎II/环II区域更一致。 一个较小的RNA已经被纯化，它对应于茎II， 用UUCG四环代替loopII，简化词干分配 共振 这个RNA间接证实了 为了与型号B保持一致， 安排使用四环结，并观察到一个几乎 相同的亚氨基光谱。 Homeland 2D 1H数据表明， 与模型B相同的碱基配对排列，但受到以下因素的限制 在1H光谱中的简并性。 这些RNA的同位素富集 将是必不可少的更完整的任务所需的 确定高分辨率结构。 其中包括完整的 随机同位素富集的RNA在15 N和13 C，和选择性 特定核苷酸的富集。

There are three classes of recoding: ribosomal frameshifting, stop-codon readthrough, and bypass of internal mRNA region. Recoding instructions may include an RNA structure within the mRNA. These structures can kinetically alter the mRNA passage through the ribosome, can interface with the ribosome directly, and can bind proteins or other co-factors and bring them into place to direct the recoding event. Within the universal code, the UGA codon signals the termination of protein synthesis. This event can be circumvented in three ways, including co-translational insertion of selenocysteine at this triplet (a special case of readthrough). For selenocysteine insertion at stop codons in eukaryotes, there are four requirements: (1) two enzymes essential for the synthesis of the selenocysteine amino acid; (2) a special tRNA; (3) an RNA stem-loop structure, termed the selnocysteine insertion element (SECIS), is required in the 3'-UTR of the mRNAs and; (4) a protein which may act as the homologue to the prokaryotic SelB protein (SBP) is thought to bind the SECIS and program the ribosome for selenocysteine insertion. An NMR structural investigation of this structure will help to understand how an element in the 3'-UTR of a specific mRNA "informs" ribosomes translating that same mRNA that all UGA codons, with the exception of the terminal codon, should specify selenocysteine insertion. Two models of the SECIS secondary structure are available. Both models identify two stems separated by an internal loop, in addition to an apical loop. Phylogeny identifies three consecutive adenosines in loop II, and a run or 4 base-pairs in the "core" region that are essential for the function of SECIS. The base pairing register of stem II differs in the two models by one nucleotide including two tandem G-As which are proposed to be formed using N6-A to the N3-G, and a N7-A to N2-G H-bonds, allowing room for the restricted geometry of the U-U basepairs that flank the tandem. These intriguing helical arrangements are ideal sites for specific recognition of the SelB homologue (SBP) protein for eukaryotic selnocysteine insertion. A43 nucleotide RNA corresponding to stemII/loopII has been produced for NMR studies using runoff T7 transcription in conjunction with double ribozyme cleavage to produce homogeneous RNAs. Simino resonances in the 1D 1H spectra of this RNA lie within the chemical shift range expected for G-A base pairs. This attribute strongly suggests model B to be more consistent with the stemII/loopII region of this SECIS RNA. A smaller RNA has been purified, which corresponds to stemII with a UUCG tetraloop replacing loopII, to simplify the assignment of stem resonances. This RNA has indirectly confirmed the base pairing register to be consistent with model B, by forcing this stem arrangement using the tetra-loop junction, and observing a nearly identical imino spectrum. Homonuclear 2D 1H data have suggested the same base-pairing arrangement of model B, but have been limited by degeneracies in the 1H spectra. Isotopic enrichment of these RNAs will be essential for more complete assignments required for the determination of a high resolution structure. These include complete random isotopic enrichment of RNAs in both 15N and 13C, and selective enrichment of specific nucleotides.