Structure-function relationships of archaeal Cbf5 during in vivo RNA-guided pseudouridylation.
Structure-function relationships of archaeal Cbf5 during in vivo RNA-guided pseudouridylation.
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DOI：
10.1261/rna.057547.116
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发表时间：
2016-10
期刊：
影响因子：
--
通讯作者：
Gupta R
中科院分区：
文献类型：
--
作者：
Majumder M;Bosmeny MS;Gupta R
在真核生物和真核生物中，除了仅蛋白质的假尿苷（pseudouridine，pD）脱氢酶外，含有一种指导RNA和四种蛋白质的复合物也可以产生pD。cbf 5蛋白是复合物中的cbf 5合酶。以前，我们表明，在位置1940，1942，和2605的Haloferax volcanii 23 S rRNA的cbf 5缺失的菌株中是不存在的，和质粒携带的拷贝cbf 5可以拯救合成这些cbf 5的。基于已发表的与其他蛋白质和RNA复合的古细菌Cbf 5结构的报道，我们鉴定了H. volcanii Cbf 5，预期在假尿苷化中发挥重要作用。我们对这些结构进行了突变，并确定了它们在体内条件下对三个rRNA位置的β-内酰胺酶产生的影响。在催化结构域中的几个残基和拇指环中的某些残基的突变要么废除了ESTA或产生部分修饰;后者表明ESTA形成的速率较慢。Cbf 5中天冬氨酸酶的通用催化天冬氨酸可以被谷氨酸取代。不需要Cbf 5和TruB共有的保守组氨酸，但Cbf 5的另一个保守组氨酸是体内RNA引导的转录因子形成所需的。我们还鉴定了Cbf 5的假尿苷化活性中的先前未报道的新奇，其中指导H/ACA RNA的单个茎环用于产生两个紧密放置的β，并且Cbf 5的某些残基的突变消除了这两个β中的一个。总之，这第一个在体内研究确定了几个结构的古细菌Cbf 5蛋白是重要的RNA引导的假尿苷化活性。
In Eukarya and Archaea, in addition to protein-only pseudouridine (Ψ) synthases, complexes containing one guide RNA and four proteins can also produce Ψ. Cbf5 protein is the Ψ synthase in the complex. Previously, we showed that Ψ’s at positions 1940, 1942, and 2605 of Haloferax volcanii 23S rRNA are absent in a cbf5-deleted strain, and a plasmid-borne copy of cbf5 can rescue the synthesis of these Ψ’s. Based on published reports of the structure of archaeal Cbf5 complexed with other proteins and RNAs, we identified several potential residues and structures in H. volcanii Cbf5, which were expected to play important roles in pseudouridylation. We mutated these structures and determined their effects on Ψ production at the three rRNA positions under in vivo conditions. Mutations of several residues in the catalytic domain and certain residues in the thumb loop either abolished Ψ’s or produced partial modification; the latter indicates a slower rate of Ψ formation. The universal catalytic aspartate of Ψ synthases could be replaced by glutamate in Cbf5. A conserved histidine, which is common to Cbf5 and TruB is not needed, but another conserved histidine of Cbf5 is required for the in vivo RNA-guided Ψ formation. We also identified a previously unreported novelty in the pseudouridylation activity of Cbf5 where a single stem–loop of a guide H/ACA RNA is used to produce two closely placed Ψ’s and mutations of certain residues of Cbf5 abolished one of these two Ψ’s. In summary, this first in vivo study identifies several structures of an archaeal Cbf5 protein that are important for its RNA-guided pseudouridylation activity.
影响因子：
0.8
作者：
Karijolich J;Yu YT
通讯作者：
Yu YT