Cech T R, Zaug A J, Grabowski P J
Department of Chemistry, University of Colorado, Boulder 80309.
Cell. 1981 Dec;27(3 Pt 2):487-96. doi: 10.1016/0092-8674(81)90390-1.
In previous studies of transcription and splicing of the ribosomal RNA precursor in isolated Tetrahymena nuclei, we found that the intervening sequence (IVS) was excised as a unique linear RNA molecule and was subsequently cyclized. In the present work, transcription at low monovalent cation concentration is found to inhibit splicing and to lead to the accumulation of a splicing intermediate. This intermediate contains splicing activity that either is tightly bound to the RNA or is part of the RNA molecule itself. The intermediate is able to complete the excision of the IVS when it is incubated with a monovalent cation (75 mM (NH4)2SO4), a divalent cation (5-10 mM MgCl2) and a guanosine compound (1 microM GTP, GDP, GMP or guanosine). ATP, UTP, CTP and guanosine compounds without 2' and 3' hydroxyl groups are inactive in causing excision of the IVS. Accurate excision of the IVS, cyclization of the IVS and (apparently) ligation of the 26S rRNA sequences bordering the IVS all take place under these conditions, suggesting that a single activity is responsible for all three reactions. During excision of the IVS, the 3' hydroxyl of the guanosine moiety becomes linked to the 5' end of the IVS RNA via a normal phosphodiester bond. When GTP is used to drive the reaction, it is added intact without hydrolysis. Based on these results, we propose that Tetrahymena pre-rRNA splicing occurs by a phosphoester transferase mechanism. According to this model, the guanosine cofactor provides the free 3' hydroxyl necessary to initiate a series of three transfers that results in splicing of the pre-rRNA and cyclization of the excised IVS.
在之前对分离的四膜虫细胞核中核糖体RNA前体的转录和剪接研究中，我们发现间隔序列（IVS）作为一个独特的线性RNA分子被切除，随后环化。在本研究中，发现在低单价阳离子浓度下转录会抑制剪接并导致剪接中间体的积累。该中间体含有紧密结合于RNA或本身就是RNA分子一部分的剪接活性。当该中间体与单价阳离子（75 mM硫酸铵）、二价阳离子（5 - 10 mM氯化镁）和鸟苷化合物（1 μM GTP、GDP、GMP或鸟苷）一起孵育时，能够完成IVS的切除。没有2'和3'羟基的ATP、UTP、CTP和鸟苷化合物在促使IVS切除方面无活性。在这些条件下，IVS的精确切除、IVS的环化以及与IVS相邻的26S rRNA序列的（明显）连接均会发生，这表明单一活性负责所有这三个反应。在IVS切除过程中，鸟苷部分的3'羟基通过正常的磷酸二酯键与IVS RNA的5'端相连。当使用GTP驱动反应时，它完整添加而不水解。基于这些结果，我们提出四膜虫前体rRNA剪接通过磷酸酯转移酶机制发生。根据该模型，鸟苷辅因子提供启动一系列三次转移所需的游离3'羟基，这导致前体rRNA的剪接和切除的IVS的环化。
