Substitutions of Thr30 provide mechanistic insight into tryptophan-mediated activation of TRAP binding to RNA.
Substitutions of Thr30 provide mechanistic insight into tryptophan-mediated activation of TRAP binding to RNA.
复制标题
THR30的取代提供了对色氨酸介导的陷阱结合与RNA的激活的机械洞察力。
DOI：
10.1093/nar/gkl383
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发表时间：
2006
影响因子：
14.9
通讯作者：
Gollnick, Paul
中科院分区：
文献类型：
--
作者：
Payal, Vandana;Gollnick, Paul
TRAP是一种11亚基RNA结合蛋白，调节枯草芽孢杆菌中参与色氨酸生物合成和运输的基因表达。TRAP通过在邻近亚基形成的口袋中结合多达11个l-色氨酸分子来激活与RNA的结合。色氨酸结合激活TRAP的确切机制尚不清楚。Thr30在色氨酸结合袋中。一个Thr30被Val （T30V）取代的TRAP突变体不结合色氨酸，而是组成性地结合RNA，这表明Thr30在激活机制中起关键作用。我们已经研究了Thr30的其他取代的影响。在残基30处具有小β支化脂肪侧链的TRAP蛋白可组成性地结合RNA，而具有小极性侧链的TRAP蛋白则显示色氨酸依赖性RNA结合。一些突变蛋白表现出由色氨酸增强的组成性RNA结合。虽然色氨酸和RNA结合位点在TRAP上是不同的，并且相隔约7.5 Å，但与结合RNA相互作用的残基的几个替换恢复了色氨酸与T30V TRAP的结合。这些观察结果支持了TRAP在色氨酸和RNA结合位点之间传递信息的构象变化的假设。
TRAP is an 11 subunit RNA binding protein that regulates expression of genes involved in tryptophan biosynthesis and transport in Bacillus subtilis. TRAP is activated to bind RNA by binding up to 11 molecules of l-tryptophan in pockets formed by adjacent subunits. The precise mechanism by which tryptophan binding activates TRAP is not known. Thr30 is in the tryptophan binding pocket. A TRAP mutant in which Thr30 is substituted with Val (T30V) does not bind tryptophan but binds RNA constitutively, suggesting that Thr30 plays a key role in the activation mechanism. We have examined the effects of other substitutions of Thr30. TRAP proteins with small β-branched aliphatic side chains at residue 30 bind RNA constitutively, whereas those with a small polar side chain show tryptophan-dependent RNA binding. Several mutant proteins exhibited constitutive RNA binding that was enhanced by tryptophan. Although the tryptophan and RNA binding sites on TRAP are distinct and are separated by ∼7.5 Å, several substitutions of residues that interact with the bound RNA restored tryptophan binding to T30V TRAP. These observations support the hypothesis that conformational changes in TRAP relay information between the tryptophan and RNA binding sites of the protein.
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