Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan.
Nucleic Acids Res. 2011 Dec;39(22):9695-704. doi: 10.1093/nar/gkr692. Epub 2011 Aug 31.
tRNA splicing endonucleases, essential enzymes found in Archaea and Eukaryotes, are involved in the processing of pre-tRNA molecules. In Archaea, three types of splicing endonuclease [homotetrameric: α(4), homodimeric: α(2), and heterotetrameric: (αβ)(2)] have been identified, each representing different substrate specificity during the tRNA intron cleavage. Here, we discovered a fourth type of archaeal tRNA splicing endonuclease (ε(2)) in the genome of the acidophilic archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2 and its closely related species, ARMAN-1. The enzyme consists of two duplicated catalytic units and one structural unit encoded on a single gene, representing a novel three-unit architecture. Homodimeric formation was confirmed by cross-linking assay, and site-directed mutagenesis determined that the conserved L10-pocket interaction between catalytic and structural unit is necessary for the assembly. A tRNA splicing assay reveal that ε(2) endonuclease cleaves both canonical and non-canonical bulge-helix-bulge motifs, similar to that of (αβ)(2) endonuclease. Unlike other ARMAN and Euryarchaeota, tRNAs found in ARMAN-2 are highly disrupted by introns at various positions, which again resemble the properties of archaeal species with (αβ)(2) endonuclease. Thus, the discovery of ε(2) endonuclease in an archaeon deeply branched within Euryarchaeota represents a new example of the coevolution of tRNA and their processing enzymes.
tRNA 剪接内切酶是古菌和真核生物中必需的酶，参与前 tRNA 分子的加工。在古菌中，已经鉴定出三种剪接内切酶[同源四聚体：α(4)、同二聚体：α(2)和异四聚体：(αβ)(2)]，它们在 tRNA 内含子切割过程中代表不同的底物特异性。在这里，我们在嗜酸古菌 Candidatus Micrarchaeum acidiphilum 的基因组中发现了第四种古菌 tRNA 剪接内切酶（ε(2)），称为 ARMAN-2 及其密切相关的物种 ARMAN-1。该酶由两个重复的催化单元和一个结构单元组成，由单个基因编码，代表一种新的三单元结构。通过交联实验证实了同二聚体的形成，定点突变确定了催化单元和结构单元之间保守的 L10 袋相互作用对于组装是必需的。tRNA 剪接实验表明，ε(2)内切酶切割规范和非规范的凸起-螺旋-凸起模体，类似于(αβ)(2)内切酶。与其他 ARMAN 和广古菌不同，在 ARMAN-2 中发现的 tRNA 在内含子的各种位置高度被打断，这再次类似于具有(αβ)(2)内切酶的古菌的特性。因此，在深嵌于广古菌中的古菌中发现 ε(2)内切酶代表了 tRNA 及其加工酶共同进化的一个新例子。
