A tyrosyl-tRNA synthetase suppresses structural defects in the two major helical domains of the group I intron catalytic core.
A tyrosyl-tRNA synthetase suppresses structural defects in the two major helical domains of the group I intron catalytic core.
复制标题
酪氨酰-tRNA 合成酶抑制 I 组内含子催化核心的两个主要螺旋结构域的结构缺陷。
DOI：
10.1006/jmbi.1996.0501
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Lambowitz,AM
中科院分区：
文献类型：
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作者：
Myers,CA;Wallweber,GJ;Rennard,R;Kemel,Y;Caprara,MG;Mohr,G;Lambowitz,AM
粗糙脉孢菌线粒体酪氨酰-tRNA合成酶（CYT-18蛋白）通过促进内含子RNA的催化活性结构的形成而在剪接I组内含子中起作用。I组内含子催化核心被认为由两个延伸的螺旋结构域组成，一个由P5、P4、P6和P6 a（P4-P6结构域）的同轴堆叠形成，另一个由P8、P3、P7和P9（P3-P9结构域）组成。为了研究CYT-18如何稳定活性RNA结构，我们使用基于噬菌体T4 tdintron的大肠杆菌遗传学测定来系统地测试CYT-18补偿催化核心的三个关键区域中的结构缺陷的能力：J3/4和J6/7，与P4-P6结构域形成三螺旋支架结构的部分的连接区域，和P7，形成鸟苷结合位点的长距离碱基配对相互作用，是P3-P9结构域的一部分。我们的研究结果表明，CYT-18可以抑制许多破坏J3/4和J6/7核苷酸三重相互作用的突变，以及破坏P7中碱基配对的突变。CYT-18抑制了在所有测试位置的遗传学保守核苷酸残基的突变，除了在鸟苷结合位点的普遍保守的G-残基。用选定的突变内含子进行的结构作图实验表明，CYT-18抑制性J3/4突变主要损害P4-P6结构域的折叠，而J6/7突变在不同程度上损害P4-P6和P3-P9结构域的折叠。P7突变损害了P7和P3的形成，从而严重破坏了P3-P9结构域。P7突变也损害了P3的形成，这一发现提供了证据，表明这两种长距离配对的形成在tdintron中是相互依赖的。与以前的工作一起考虑，CYT-18抑制突变的性质支持这样一种模型，即CYT-18帮助组装P4-P6结构域，然后稳定催化核心的两个主要螺旋结构域，使其处于正确的相对方向，以形成内含子的活性位点。
TheNeurospora crassamitochondrial tyrosyl-tRNA synthetase, the CYT-18 protein, functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA. The group I intron catalytic core is thought to consist of two extended helical domains, one formed by coaxial stacking of P5, P4, P6, and P6a (P4-P6 domain) and the other consisting of P8, P3, P7, and P9 (P3-P9 domain). To investigate how CYT-18 stabilizes the active RNA structure, we used anEscherichia coligenetic assay based on the phage T4tdintron to systematically test the ability of CYT-18 to compensate for structural defects in three key regions of the catalytic core: J3/4 and J6/7, connecting regions that form parts of the triple-helical-scaffold structure with the P4-P6 domain, and P7, a long- range base-pairing interaction that forms the guanosine-binding site and is part of the P3-P9 domain. Our results show that CYT-18 can suppress numerous mutations that disrupt the J3/4 and J6/7 nucleotide-triple interactions, as well as mutations that disrupt base-pairing in P7. CYT-18 suppressed mutations of phylogenetically conserved nucleotide residues at all positions tested, except for the universally conserved G-residue at the guanosine-binding site. Structure mapping experiments with selected mutant introns showed that the CYT-18-suppressible J3/4 mutations primarily impaired folding of the P4-P6 domain, while the J6/7 mutations impaired folding of both the P4-P6 and P3-P9 domains to various degrees. The P7 mutations impaired the formation of both P7 and P3, thereby grossly disrupting the P3-P9 domain. The finding that the P7 mutations also impaired formation of P3 provides evidence that the formation of these two long-range pairings is interdependent in thetdintron. Considered together with previous work, the nature of mutations suppressed by CYT-18 supports a model in which CYT-18 helps assemble the P4-P6 domain and then stabilizes the two major helical domains of the catalytic core in the correct relative orientation to form the intron's active site.