Molecular dissection of the Mycobacterium tuberculosis RecA intein:: design of a minimal intein and of a trans-splicing system involving two intein fragments
Molecular dissection of the Mycobacterium tuberculosis RecA intein:: design of a minimal intein and of a trans-splicing system involving two intein fragments
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DOI：
10.1016/s0378-1119(97)00624-0
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发表时间：
1998-01-30
期刊：
影响因子：
3.5
通讯作者：
Paulus, H
中科院分区：
文献类型：
--
作者：
Shingledecker, K;Jiang, SQ;Paulus, H
大多数蛋白质剪接元件（内含肽）既作为蛋白质剪接的催化剂，又作为归巢核酸内切酶。为了确定蛋白质剪接所必需的内含肽结构域，对结核分枝杆菌recA基因中嵌入的内含肽序列进行了遗传解剖。在用质粒转化的大肠杆菌中研究了内含肽的各种修饰对介导剪接能力的影响，其中RecA内含肽的编码序列被插入到大肠杆菌编码区之间的读框内。大肠杆菌麦芽糖结合蛋白和含有六聚组氨酸序列的多肽分别作为N-和C-外显蛋白。RecA内含肽的一种类型的遗传改变涉及编码229个氨基酸（aa）的中心区域的缺失，代表整个归巢核酸内切酶同源结构域。剩余的内含肽（211个氨基酸加上一个十一肽间隔）能够促进蛋白质剪接的有效野生型内含肽，表明归巢核酸内切酶结构域在蛋白质剪接过程中不起作用，蛋白质剪接活性中心仅限于N和C-末端的内含肽，小于110个氨基酸。另一种类型的改变涉及将重叠的翻译终止和起始密码子框内引入内含肽编码区。修饰的RecA内含肽，虽然合成为两个单独的组分，但仍然可以介导蛋白质剪接，表明N-和C-末端蛋白质剪接结构域可以以足够的亲和力和特异性相互作用，以允许蛋白质剪接以反式发生。当归巢核酸内切酶结构域完全缺失时，反式剪接的效率大大增强，使得相互作用的N-和C-末端蛋白质剪接结构域的长度增加。末端内含肽片段仅为约110个氨基酸。(C)1998年Elsevier Science B.V.
Most protein-splicing elements (inteins) function both as catalysts of protein splicing and as homing endonucleases. In order to identify the domains of inteins that are essential for protein splicing, the intein sequence embedded in the recA gene of Mycobacterium tuberculosis was genetically dissected. The effect of various modifications of the intein on the ability to mediate splicing was studied in Escherichia coli transformed with plasmids is which the coding sequence for the RecA intein was inserted in-frame between coding regions for the E. coli maltose-binding protein and a polypeptide containing a hexahistidine sequence as the N- and C-exteins, respectively. One type of genetic alteration of the RecA intein involved deletion of the the central region encoding 229 amino acids (aa), representing the entire homing endonuclease homology domain. The residual intein (211 aa plus an undecapeptide spacer) was able to promote protein splicing as efficiently as the wild-type intein, indicating that the homing endonuclease domain plays no role in the protein-splicing process and that the protein-splicing active center is confined to the Nand C-terminal segments of the intein, less than 110 aa each. Another type of alteration involved the introduction of overlapping translation termination and initiation codons in-frame into the intein coding region. The modified RecA intein, although synthesized as two separate components, could nevertheless mediate protein splicing, indicating that the N- and C-terminal protein-splicing domains can interact with sufficient affinity and specificity to allow protein-splicing to occur in trans. The efficiency of trans-splicing was much enhanced when the homing endonuclease domain was entirely deleted so that the length of the interacting N- and C-terminal intein fragments was only about 110 aa each. (C) 1998 Elsevier Science B.V.