Crystal structure and structure-based mutational analyses of RNase hill from Bacillus stearothermophilus:: A new type 2 RNase H with TBP-like substrate-binding domain at the N terminus
Crystal structure and structure-based mutational analyses of RNase hill from Bacillus stearothermophilus:: A new type 2 RNase H with TBP-like substrate-binding domain at the N terminus
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DOI：
10.1016/j.jmb.2005.11.017
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发表时间：
2006-02-10
影响因子：
5.6
通讯作者：
Kanaya, S
中科院分区：
文献类型：
--
作者：
Chon, H;Matsumura, H;Kanaya, S
来自中度嗜热芽孢杆菌的核糖核酸酶HIII（Bst-RNase HIII）是2型RNase H，但与另一种2型RNase H（RNase HII）显示差的氨基酸序列同一性。它由310个氨基酸残基组成，并作为单体发挥作用。Bst-RNase HIII具有一个功能未知的大N-末端延伸和一个独特的活性位点基序（DEDE），这两个都是RNase HIII共有的特征。为了理解这些N-末端延伸和活性位点残基的作用，在2.1-2.6埃分辨率下以无金属和金属结合形式测定了Bst-RNase HIII的晶体结构。根据这些结构，Bst-RNase HIII由N-末端结构域和C-末端RNase H结构域组成。的N和C-末端结构域的结构相似的TATA盒结合蛋白和古细菌RNA酶HII，分别。四个保守活性位点残基的空间构型与其他1型和2型RNase H非常相似。单个Mn和Mg离子与Asp 97、Glu 98和Asp 202配位，其分别对应于大肠杆菌RNase HI的Asp 10、Glu 48和Asp 70。突变研究表明，这些残基中的任何一个被Ala取代都会导致酶活性的极大降低。过量生产，纯化和表征的Bst-RNase HIII衍生物与N和/或C-末端截短表明，N-末端结构域和C-末端螺旋参与底物结合，但前者比后者更有助于底物结合。(c)2005爱思唯尔有限公司保留所有权利。
Ribonuclease HIII (Bst-RNase HIII) from the moderate thermophile Bacillus stearothermophilus is a type 2 RNase H but shows poor amino acid sequence identity with another type 2 RNase H, RNase HII. It is composed of 310 amino acid residues and acts as a monomer. Bst-RNase HIII has a large N-terminal extension with unknown function and a unique active-site motif (DEDE), both of which are characteristics common to RNases HIII. To understand the role of these N-terminal extension and active-site residues, the crystal structure of Bst-RNase HIII was determined in both metal-free and metal-bound forms at 2.1-2.6 angstrom resolutions. According to these structures, Bst-RNase HIII consists of the N-terminal domain and C-terminal RNase H domain. The structures of the N and C-terminal domains were similar to those of TATA-box binding proteins and archaeal RNases HII, respectively. The steric configurations of the four conserved active-site residues were very similar to those of other type 1 and type 2 RNases H. Single Mn and Mg ions were coordinated with Asp97, Glu98, and Asp202, which correspond to Asp10, Glu48, and Asp70 of Escherichia coli RNase HI, respectively. The mutational studies indicated that the replacement of either one of these residues with Ala resulted in a great reduction of the enzymatic activity. Overproduction, purification, and characterization of the Bst-RNase HIII derivatives with N and/or C-terminal truncations indicated that the N-terminal domain and C-terminal helix are involved in substrate binding, but the former contributes to substrate binding more greatly than the latter. (c) 2005 Elsevier Ltd. All rights reserved.