Involvement of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing. A new method for purifying the protein and characterization of physical and enzymatic properties pertinent to splicing.
Involvement of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing. A new method for purifying the protein and characterization of physical and enzymatic properties pertinent to splicing.
复制标题
脉孢菌线粒体酪氨酰-tRNA 合成酶参与 RNA 剪接。
DOI：
10.1021/bi00004a022
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Lambowitz,AM
中科院分区：
文献类型：
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作者：
Saldanha,RJ;Patel,SS;Surendran,R;Lee,JC;Lambowitz,AM
摘要：脉孢菌CYT-18蛋白是线粒体酪氨酰-tRNA合成酶，在I组内含子的剪接中起作用。在这里，细菌表达的CYT-18蛋白，纯化的一个新的程序，涉及聚乙烯亚胺沉淀，以消除紧密结合的核酸，被用来表征有关RNA剪接的属性。超电泳和其他方法分析表明，CYT-18蛋白是一个不对称的同源二聚体。测得的摩擦比f/f0= 1-55，对应于长椭球体的轴比10或扁椭球体的轴比12。与细菌TyrRS一样，CYT-18蛋白表现出半位点反应性，每个同二聚体具有一个用于酪氨酰化和RNA剪接的活性位点。CYT-18的剪接活性在氨酰化反应中使用的浓度下不受氨酰化底物的影响，而TyrRS活性被生理浓度的剪接辅因子GTP以及CTP或UTP或低浓度的I组内含子RNA抑制。动力学测量表明CYT-18与I组内含子底物的结合是两步过程，初始双分子步骤接近扩散限制（3.24±0.03 × 107 M_1 s_1），随后是较慢的构象变化（0.54±0.07 s_1）。CYT-18结合后，剪接发生在0.0025 s-1的速率，在6倍的四膜虫大rRNA内含子在体外的自我剪接的速率。由koff/kon计算的CYT-18蛋白和I组内含子底物之间的复合物的Kd <0.3pM，显著低于通过假定的平衡测量确定的Kd [Guo，Q.，& Lambowitz，AM（1992）Genes Dev. 6，1357-1372]，由于这种紧密结合，CYT-18蛋白质在体外剪接反应中以化学计量方式起作用，这是由于其与切除的内含子RNA的解离极其缓慢。CYT-18蛋白与内含子RNA的非常紧密的结合提高了在体内将蛋白从切除的内含子解离的特定机制存在的可能性。（mt TyrRS）1由核基因cyt-18编码，在链孢菌线粒体中参与mt大rRNA内含子和其他I组内含子的剪接（柯林斯和兰博维茨，1985年;艾金斯和兰博维茨，1987年）。CYT-
Revised Manuscript Received October 5, 1994® abstract: The NeurosporaCYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in the splicing of group I introns. Here, bacterially expressed CYT-18 protein, purified by a new procedure involving polyethyleneimine precipitation to remove tightly bound nucleic acids, was used to characterize properties pertinent to RNA splicing. Analytical ultracentrifugation and other methods showed that the CYT-18 protein is an asymmetric homodimer. The measured frictional ratio, f/f0= 1-55, corresponds to an axial ratio of 10 for a prolate ellipsoid or 12 for an oblate ellipsoid. Like bacterial TyrRSs, the CYT-18 protein exhibits half-sites reactivity, each homodimer having one active site for tyrosyladenylation and RNA splicing. The splicing activity of CYT-18 was unaffected by aminoacylation substrates at concentrations used in aminoacylation reactions, whereas the TyrRSactivity was inhibited by physiological concentrations of the splicing cofactor GTP, as well as CTP or UTP, or by low concentrations of a group I intron RNA. Kinetic measurements suggest that the binding of CYT-18 to a group I intron substrate is a two-step process, with an initial bimolecular step that is close to diffusion limited (3.24±0.03 x 107 M_1 s_1) followed by a slower conformational change (0.54±0.07 s_1). After CYT-18binding, splicing occurs at a rate of 0.0025 s-1, within 6-fold of the rate of self-splicing of the Tetrahymena large rRNA intron in vitro. The Kd for the complex between the CYT-18 protein and a group I intron substrate, calculated from k0ff/k0n, was< 0.3 pM, substantially lower than determined by presumed equilibrium measurements [Guo, Q., & Lambowitz, AM (1992) Genes Dev. 6, 1357—1372], As a result of this tight binding, the CYT-18 protein functions stoichiometrically in in vitro splicing reactionsdue to its extremely slow dissociation from the excised intron RNA. The very tight binding of the CYT-18 protein to the intron RNA raises the possibility that specific mechanisms exist for dissociating the protein from the excised intron in vivo.The Neurospora mitochondrialtyrosyl-tRNA synthetase (mt TyrRS), 1 which is encoded by the nuclear gene cyt-18, functions in the splicing of the mt large rRNA intron and other group I introns in Neurospora mitochondria (Collins & Lambowitz, 1985; Akins & Lambowitz, 1987). The CYT-