Characterization of a trp RNA-binding attenuation protein (TRAP) mutant with tryptophan independent RNA binding activity
Characterization of a trp RNA-binding attenuation protein (TRAP) mutant with tryptophan independent RNA binding activity
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DOI：
10.1016/j.jmb.2003.11.002
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发表时间：
2004-01-16
影响因子：
5.6
通讯作者：
Gollnick, P
中科院分区：
文献类型：
--
作者：
Li, PTX;Gollnick, P
TRAP（trp RNA结合衰减蛋白）是11个亚基的RNA结合蛋白，其响应于细胞内色氨酸浓度的变化而调节枯草芽孢杆菌中参与色氨酸代谢（trp）的基因的表达。当通过结合多达11个色氨酸残基而被激活时，TRAP结合几个trp基因的mRNA并下调其表达。最近，发现TRAP突变体在色氨酸不存在的情况下结合RNA。在该突变蛋白中，Thr 30（其是聚糖结合位点的一部分）被瓦尔取代（T30 V）。我们比较了T30 V和野生型（WT）TRAP的RNA结合特性，以及一系列含有WT和T30 V TRAP亚基混合物的异源11聚体。T30 V和WT TRAP与RNA的相互作用之间最显著的差异是T30 V TRAP的亲和力更依赖于离子强度。异源11聚体的分析使我们能够研究亚基如何在11聚体内相互作用，从而与色氨酸或RNA结合。我们的数据表明，个别亚基保留的性质类似于观察到的，当他们在homo-11-mer和个别G/UAG三联体内的RNA结合到TRAP不同。(C)2003爱思唯尔有限公司。保留所有权利。
TRAP (trp RNA-binding attenuation protein) is an 11 subunit RNA-binding protein that regulates expression of genes involved in tryptophan metabolism (trp) in Bacillus subtilis in response to changes in intracellular tryptophan concentration. When activated by binding up to 11 tryptophan residues, TRAP binds to the mRNAs of several trp genes and downregulates their expression. Recently, a TRAP mutant was found that binds RNA in the absence of tryptophan. In this mutant protein, Thr30, which is part of the tryptophan-binding site, is replaced with Val (T30V). We have compared the RNA-binding properties of T30V and wild-type (WT) TRAP, as well as of a series of hetero-11-mers containing mixtures of WT and T30V TRAP subunits. The most significant difference between the interaction of T30V and WT TRAP with RNA is that the affinity of T30V TRAP is more dependent on ionic strength. Analysis of the hetero-11-mers allowed us to examine how subunits interact within an 11-mer with regard to binding to tryptophan or RNA. Our data suggest that individual subunits retain properties similar to those observed when they are in homo-11-mers and that individual G/UAG triplets within the RNA an bind to TRAP differently. (C) 2003 Elsevier Ltd. All rights reserved.