Alanine scanning mutagenesis of anti-TRAP (AT) reveals residues involved in binding to TRAP
Alanine scanning mutagenesis of anti-TRAP (AT) reveals residues involved in binding to TRAP
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DOI：
10.1016/j.jmb.2008.02.015
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发表时间：
2008-04-11
影响因子：
5.6
通讯作者：
Gollnick, Paul
中科院分区：
文献类型：
--
作者：
Chen, Yanling;Gollnick, Paul
Trp RNA结合衰减蛋白（TRAP）调节色氨酸生物合成（trp）基因的表达，以响应许多革兰氏阳性菌中游离1-色氨酸细胞内水平的变化。当通过结合色氨酸激活时，TRAP结合参与色氨酸代谢的几个基因的mRNA，并下调这些基因的转录或翻译。抗TRAP（AT）是TRAP的拮抗剂，其结合至聚糖活化的TRAP并阻止其结合至其RNA靶标，从而上调trp基因表达。晶体结构表明AT是一个锥形的三聚体（AT（3）），三个亚基的N-末端残基在圆锥的顶点组装，并且这些三聚体可以进一步组装成十二聚体（AT（12））结构。使用丙氨酸扫描诱变，我们发现了四个残基，都位于AT（3）的“顶部”区域，这是必不可少的结合TRAP。荧光标记实验进一步表明，AT的顶部区域在AT-TRAP复合物中与TRAP紧密并列。体内研究证实了AT顶部的这些残基在调节TRAP介导的基因调控中的重要性。(C)2008爱思唯尔有限公司保留所有权利。
The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic (trp) genes in response to changes in intracellular levels of free 1-tryptophan in many Gram-positive bacteria. When activated by binding tryptophan, TRAP binds to the mRNAs of several genes involved in tryptophan metabolism, and down-regulates transcription or translation of these genes. Anti-TRAP (AT) is an antagonist of TRAP that binds to tryptophan-activated TRAP and prevents it from binding to its RNA targets, and thereby up-regulates trp gene expression. The crystal structure shows that AT is a cone-shaped trimer (AT(3)) with the N-terminal residues of the three subunits assembled at the apex of the cone and that these trimers can further assemble into a dodecameric (AT(12)) structure. Using alanine-scanning mutagenesis we found four residues, all located on the "top" region of AT(3), that are essential for binding to TRAP. Fluorescent labeling experiments further suggest that the top region of AT is in close juxtaposition to TRAP in the AT-TRAP complex. In vivo studies confirmed the importance of these residues on the top of AT in regulating TRAP mediated gene regulation. (C) 2008 Elsevier Ltd. All rights reserved.