Creating hetero-11-mers composed of wild-type and mutant subunits to study RNA binding to TRAP
Creating hetero-11-mers composed of wild-type and mutant subunits to study RNA binding to TRAP
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DOI：
10.1074/jbc.m110860200
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发表时间：
2002-04-05
影响因子：
4.8
通讯作者：
Gollnick, P
中科院分区：
文献类型：
--
作者：
Li, PTX;Scott, DJ;Gollnick, P
TRAP（(t) 下的 rp (R) 下的 NA 结合 (a) 下的条衰减 (p) 下的条蛋白）是一种 RNA 结合蛋白，通过与含有多个 GAG 和 UAG 重复序列的 RNA 靶标结合来调节枯草芽孢杆菌中色氨酸生物合成基因的表达。 TRAP由11个相同的亚基对称排列成环组成。蛋白质的二级结构完全由反平行、β-折叠、β-转角和环组成。我们在此表明，TRAP 11-mer 可以通过盐酸胍可逆地变性为未折叠单体。去除变性剂可使蛋白质自发复性为功能齐全的 11 聚体。基于这一发现，我们开发了一种亚基混合方法，通过变性然后亚基混合复性将野生型和突变亚基杂交成异聚体 11 聚体。该方法允许研究蛋白质-配体相互作用（例如 RNA 结合）中的亚基协同性。我们的数据进一步支持和扩展了先前提出的 RNA 与 TRAP 结合的两步模型，表明结合的起始需要蛋白质中至少一个完全活性的亚基与 RNA 中一个完全功能的重复序列相结合。起始复合物将 RNA 束缚在蛋白质上，从而允许与其余重复序列协同相互作用。
TRAP ((t) under bar rp (R) under bar NA-binding (a) under bar ttenuation (p) under bar rotein) is an RNA-binding protein that regulates expression of the tryptophan biosynthetic genes in Bacillus subtilis by binding to RNA targets that contain multiple GAG and UAG repeats. TRAP is composed of 11 identical subunits arranged symmetrically in a ring. The secondary structure of the protein consists entirely of antiparallel,beta-sheets, beta-turns, and loops. We show here that the TRAP 11-mer can be reversibly denatured into unfolded monomers by guanidine hydrochloride. Removing the denaturant allows the protein to spontaneously renature into fully functional 11-mers. Based on this finding, we developed a subunit mixing method to hybridize wild-type and mutant subunits into heteromeric 11-mers by denaturation followed by subunit mixing renaturation. This method allows the study of subunit cooperativity in protein-ligand interaction such as RNA binding. Our data further support and extend the previously proposed two-step model for RNA binding to TRAP by showing that the initiation of binding requires at least one fully active subunit in the protein combined with one fully functional repeat in the RNA. The initiation complex tethers the RNA on the protein, thus allowing cooperative interaction with the remainder of the repeats.