Kinetic and thermodynamic analysis of the interaction between TRAP (trp RNA-binding attenuation protein) of Bacillus subtilis and trp leader RNA
Kinetic and thermodynamic analysis of the interaction between TRAP (trp RNA-binding attenuation protein) of Bacillus subtilis and trp leader RNA
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DOI：
10.1074/jbc.271.21.12269
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发表时间：
1996-05-24
影响因子：
4.8
通讯作者：
Gollnick, P
中科院分区：
文献类型：
--
作者：
Baumann, C;Otridge, J;Gollnick, P
在枯草杆菌中，色氨酸生物合成基因的表达受到一种称为TRAP（trp RNA结合衰减蛋白）的RNA结合蛋白的调节，以响应色氨酸。TRAP已被证明含有11个相同的亚基排列在一个对称的环。研究了色氨酸激活的TRAP与色氨酸前导RNA相互作用的动力学和热力学参数。甘油梯度和迁移率变动凝胶的结果表明，两个TRAP 11聚体结合到每个trp前导RNA。使用滤膜结合试验测定TRAP和含trp前导RNA残基+36至+92的RNA在1 mM L-色氨酸中的表观结合常数为8.0 +/- 1.3 x 10（9）M（-1）（Kd = 0.12 +/- 0.02 nM）（37 ℃）。K-app的温度依赖性有些出乎意料，表明相互作用的Δ H在+15.9 kcal mol（-1）时非常不利。因此，相互作用完全由+97 cal mol（-1）K-1的Δ S驱动。色氨酸激活的TRAP和trp前导RNA之间的相互作用显示了广泛的盐和pH活性曲线。最后是RNA从RNA中解离的速率。陷阱发现色氨酸三元复合物在高浓度的色氨酸（>40 μ M）中非常缓慢，但在较低的色氨酸浓度中增加。这表明色氨酸从三元复合物中的解离是RNA解离的限速步骤。
In Bacillus subtilis, expression of the tryptophan biosynthetic genes is regulated in response to tryptophan by an RNA-binding protein called TRAP (trp RNA-binding attenuation protein). TRAP has been shown to contain 11 identical subunits arranged in a symmetrical ring. Kinetic and thermodynamic parameters of the interaction between tryptophan-activated TRAP and trp leader RNA were studied. Results from glycerol gradients and mobility shift gels indicate that two TRAP 11-mers bind to each trp leader RNA. A filter binding assay was used to determine an apparent binding constant of 8.0 +/- 1.3 x 10(9) M(-1) (K-d = 0.12 +/- 0.02 nM) for TRAP and an RNA containing residues +36 to +92 of the trp leader RNA in 1 mM L-tryptophan at 37 degrees C. The temperature dependence of K-app was somewhat unexpected demonstrating that the Delta H of the interaction is highly unfavorable at +15.9 kcal mol(-1). Therefore, the interaction is completely driven by a Delta S of +97 cal mol(-1) K-1. The interaction between tryptophan-activated TRAP and trp leader RNA displayed broad salt and pH activity profiles. Finally, the rate of RNA dissociation from the RNA . TRAP . tryptophan ternary complex was found to be very slow in high concentrations of tryptophan (>40 mu M) but increased in lower tryptophan concentrations. This suggests that dissociation of tryptophan from the ternary complex is the rate-limiting step in RNA dissociation.