Structure of the guanylyltransferase domain of human mRNA capping enzyme
Structure of the guanylyltransferase domain of human mRNA capping enzyme
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DOI：
10.1073/pnas.1106610108
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发表时间：
2011-06-21
影响因子：
11.1
通讯作者：
Shatkin, Aaron J.
中科院分区：
文献类型：
--
作者：
Chu, Chun;Das, Kalyan;Shatkin, Aaron J.
鸟苷基转移酶（GTase）在向新生mRNA （pre-mRNA）共转录添加（m7）GpppN帽的三步催化过程中起核心作用。从单细胞生物到人类，5'-mRNA盖帽过程在功能和进化上都是保守的。然而，来自病毒和酵母的gtase与来自哺乳动物的gtase具有较低的氨基酸序列一致性（类似于25%），相反，来自哺乳动物的gtase具有高度保守性（类似于98%）。通过有限的蛋白水解，我们定义了人类帽盖酶残基229-567为包含最低酶活性的人类GTase （hGTase）结构域，并通过x射线晶体学确定了其结构。晶体中存在7种相关的hGTase构象态。gtp结合位点在进化和结构上是保守的。寡核苷酸/寡糖结合褶盖结构域在gtp结合位点上的位置变化提供了活性位点间隙通过旋转运动打开和关闭的快照。哺乳动物的保守表面残基模式，而不是酵母，支持了RNA聚合酶II和相关转录因子在哺乳动物中对盖帽装置的识别是高度保守的，其机制可能与酵母有所不同。hGTase结构应该有助于设计生化和分子生物学实验，以探索蛋白质：蛋白质和蛋白质：RNA相互作用，确保人类和其他哺乳动物基因转录的调节。
The enzyme guanylyltransferase (GTase) plays a central role in the three-step catalytic process of adding an (m7)GpppN cap cotranscriptionally to nascent mRNA (pre-mRNAs). The 5'-mRNA capping process is functionally and evolutionarily conserved from unicellular organisms to human. However, the GTases from viruses and yeast have low amino acid sequence identity (similar to 25%) with GTases from mammals that, in contrast, are highly conserved (similar to 98%). We have defined by limited proteolysis of human capping enzyme residues 229-567 as comprising the minimum enzymatically active human GTase (hGTase) domain and have determined the structure by X-ray crystallography. Seven related conformational states of hGTase exist in the crystal. The GTP-binding site is evolutionarily and structurally conserved. The positional variations of the oligonucleotide/oligosaccharide binding fold lid domain over the GTP-binding site provide snapshots of the opening and closing of the active site cleft through a swivel motion. The pattern of conserved surface residues in mammals, but not in yeast, supports the finding that the recognition of the capping apparatus by RNA polymerase II and associated transcription factors is highly conserved in mammals, and the mechanism may differ somewhat from that in yeast. The hGTase structure should help in the design of biochemical and molecular biology experiments to explore the protein: protein and protein: RNA interactions that ensure regulated transcription of genes in humans and other mammals.