Enzymatic and crystallographic characterization of archaeal tRNA splicing endonuclease
Enzymatic and crystallographic characterization of archaeal tRNA splicing endonuclease
复制标题
古菌 tRNA 剪接核酸内切酶的酶学和晶体学表征
DOI：
--
复制
发表时间：
2009
期刊：
影响因子：
--
通讯作者：
H. Hori
中科院分区：
文献类型：
--
作者：
Tsubasa Kitajima;A. Hirata;Chikako Iwashita;S. Yokobori;H. Hori
RNA剪接内切酶在真核生物和古细菌中是保守的，它能从真核生物的核tRNA和古细菌的所有RNA物种中去除内含子。古细菌RNA剪接内切酶的亚基组成有三种类型，即α2（某些Euryarchaea属同二聚体）、α4（其他Euryarchaea属同四聚体）和（αβ）2 （Crenarchaea属异四聚体）。在Crenarchaea中，tRNA前体（pre-tRNA）分子中的内含子不仅存在于反密码子臂中，还存在于tRNA的其他几个区域，如D-环和t -环、可变区和氨基基茎中。这些内含子具有一致的凸起-螺旋-凸起基序。古细菌RNA剪接内切酶可以去除这些非规范位点上的内含子。然而，该酶的广泛裂解位点特异性仍然是排他性的。本研究利用内含子分别位于反密码子和d环上的前trnathr （UGU）和前trnathr (CGU)，报道了来自超嗜热性Aeropyrum pernix （APE）的重组RNA剪接内切酶的裂解活性。此外，我们报道了APE RNA剪接酶在空间群P3(1)中结晶，在不对称单元中有两个或三个异源四聚体。收集了2.8 Å分辨率的x射线衍射数据集。结构测定正在进行中。
RNA splicing endonuclease, which is conserved in Eukarya and Archaea, removes introns from eukaryotic nuclear tRNA and archaeal all RNA species. There are three types of subunit composition in archaeal RNA splicing endonucleases, namely α2 (homodimer in some Euryarchaea), α4 (homotetramer in other Euryarchaea) and (αβ)2 (heterotetramer in the Crenarchaea) types. In Crenarchaea, introns in precursor tRNA (pre-tRNA) molecules are found not only in anticodon arm but also in several other regions in tRNA such as D- and T-loops, variable region, and aminoacyl stem. These introns have a consensus bulge-helix-bulge motif. Crenarchaeal RNA splicing endonuclease can remove introns in such non-canonical sites. However, the broad cleavage site-specificity of the enzyme remains exclusive. Here, we report the cleavage activity of recombinant RNA splicing endonuclease from hyperthermophilic crenarcheon Aeropyrum pernix (APE) by using the pre-tRNAThr (UGU) and pre-tRNAThr (CGU), in which introns are located in anticodon and D-loops, respectively. Furthermore we report that APE RNA splicing enzyme crystallizes in space group P3(1) with two or three heterotetramers in an asymmetric unit. An X-ray diffraction data set has been collected to 2.8 Å resolution. Structural determination is now underway.