NEW REACTIONS OF THE RIBOSOMAL-RNA PRECURSOR OF TETRAHYMENA AND THE MECHANISM OF SELF-SPLICING
NEW REACTIONS OF THE RIBOSOMAL-RNA PRECURSOR OF TETRAHYMENA AND THE MECHANISM OF SELF-SPLICING
复制标题
DOI：
10.1016/0022-2836(86)90387-6
复制
发表时间：
1986-05-05
影响因子：
5.6
通讯作者：
CECH, TR
中科院分区：
文献类型：
--
作者：
INOUE, T;SULLIVAN, FX;CECH, TR
通过用噬菌体SP 6 RNA聚合酶转录重组质粒产生的离散大小的四膜虫前rRNA的可用性，已经对自剪接反应进行了更详细的研究。确定了预测的剪接中间体，即在5“剪接位点处被鸟苷切割的产物。在分子间外显子连接反应中测试该中间体，发现其能够进行第二步剪接。这些结果和其他评价5“和3”剪接位点反应性的结果独立地表明剪接发生在两个可分离的步骤中。发现3“剪接位点对位点特异性水解敏感，留下3”羟基末端。这被解释为表明3 ''剪接位点被激活，通常用于亲核攻击，特别是用于外显子连接。还获得了在5“剪接位点特异性水解的初步证据。所有新发现的插入序列RNA介导的反应以及以前发现的反应可分为三类：（1）鸟苷在两个或三个嘧啶核苷酸后的位点上的酯交换反应（以及，作为次要反应，在其他鸟苷残基之后的位点）;（2）通过寡嘧啶或通过5“外显子的酯交换（其以C-U-C-U-C-UOH终止）;和（3）在剪接位点的特异性水解。在3“剪接位点处的反应产物之一是含有仍然连接到间插序列的5”外显子的分子。它有一个3“-末端GOH，并在插入序列内的正常环化位点和5”剪接位点都经历环化。剪接位点可以作为环化位点的发现，结合早期观察到的正常环化位点受到鸟苷单核苷酸的攻击，使我们提出，所有这些反应可能发生在同一个活性位点。易位（构象变化）将使不同的寡嘧啶序列进入鸟苷攻击的活性位点。在实验结果的基础上，在活性位点的局部结构的模型进行了描述。该模型的一个关键特征是寡嘧啶序列末端的U、间插序列中内部指导序列内的G残基和另一个G残基之间的相互作用，该G残基可以是酯交换的攻击基团或间插序列的3“-末端G。
The availability of Tetrahymena pre-rRNA of discrete size, produced by transcription of recombinant plasmids with bacteriophage SP6 RNA polymerase, has perimitted a more detailed investigation of the self-splicing reaction. The predicted splicing intermediate, the product of cleavage by goanosine at the 5'' splice site, was identified. This intermediate was tested in the intermolecular exon ligation reaction and found to be competent to undergo the second step of splicing. These results and others that evaluated the reactivity of the 5'' and 3'' splice sites independently show that splicing occurs in two separable steps. The 3'' splice site was found to be susceptible to site-specific hydrolysis leaving a 3'' hydroxyl terminus. This in interpreted as an indication that the 3'' splice site is activated for nucleophilic attack in general and for exon ligation in particular. Preliminary evidence for specific hydrolysis at the 5'' splice site was also obtained. All of the newly characterized intervening sequence RNA-mediated reactions as well as those found previously are divided into three categories: (1) transesterification by guanosine at sites following two or three pyrimidine nucleotides (and, as a minor reaction, at sites following other guanosine residues); (2) transesterification by oligopyrimidines or by the 5'' exon (which terminates with C-U-C-U-C-UOH) at the site following the 3''-terminal guanosine residue of the intervening sequence; and (3) specific hydrolysis at the splice sites. One of the products of the reactions at the 3'' splice site is a molecule that contains the 5'' exon still attached to the intervening sequence. It has a 3''-terminal GOH and undergoes cyclization both at the normal cyclization site within the intervening sequence and at the 5'' splice site. The finding that the splice site can act as a cyclization site, combined with the earlier observation that the normal cyclization site is subject to attack by guanosine mononucleotide, leads us to propose that all these reactions may be occurring in the same active site. Translocation (a conformational change) would then bring different oligopyrimidine sequences into the active site for attack by guanosine. On the basis of the experimental results, a model for the local structure at the active site is described. A key feature of the model is the interaction between the U at the end of the oligopyrimidine sequence, a G residue within the internal guide sequence in the intervening sequence, and another G residue that can be either the attacking group for transesterification or the 3''-terminal G of the intervening sequence.