Gly184 of the Escherichia coli cAMP receptor protein provides optimal context for both DNA binding and RNA polymerase interaction.
Gly184 of the Escherichia coli cAMP receptor protein provides optimal context for both DNA binding and RNA polymerase interaction.
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大肠杆菌 cAMP 受体蛋白的 Gly184 为 DNA 结合和 RNA 聚合酶相互作用提供了最佳背景。
DOI：
10.1007/s12275-017-7266-x
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Youn,Hwan
中科院分区：
文献类型：
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作者：
Hicks,MattN;Gunasekara,Sanjiva;Serate,Jose;Park,Jin;Mosharaf,Pegah;Zhou,Yue;Lee,Jin-Won;Youn,Hwan
大肠杆菌cAMP受体蛋白（CRP）利用螺旋-转角-螺旋基序与DNA结合。CRP的识别螺旋，称为F-螺旋，包括一段六个氨基酸（Arg 180，Glu 181，Thr 182，Val 183，Gly 184和Arg 185），用于直接DNA接触。Arg 180、Glu 181和Arg 185被认为是DNA结合和特异性的重要残基，但对其他残基的研究很少。在这里，我们表明，Gly 184是另一个F-螺旋残基的CRP的转录激活功能的关键。首先，在CRP位置184处重复选择甘氨酸，因为其具有提供野生型水平转录激活活性的独特能力。为了分析甘氨酸需要量，纯化了野生型CRP和突变体G184 A、G184 F、G184 S和G184 Y，并测量了它们的体外DNA结合活性。G184 A和G184 F显示DNA结合减少，这可以解释它们的低转录激活活性。而G184 S和G184 Y则表现出明显正常的DNA亲和力。因此，需要一个额外的因素来解释G184 S和G184 Y中转录激活功能的减弱，最好的解释是它们与RNA聚合酶相互作用的扰动。如本研究所示，甘氨酸是最小的氨基酸这一事实不能完全保证其适用性。我们假设Gly 184通过赋予F-螺旋构象灵活性来实现DNA结合和RNA聚合酶相互作用的双重功能。
TheEscherichia colicAMP receptor protein (CRP) utilizes the helix-turn-helix motif for DNA binding. The CRP’s recognition helix, termed F-helix, includes a stretch of six amino acids (Arg180, Glu181, Thr182, Val183, Gly184, and Arg185) for direct DNA contacts. Arg180, Glu181 and Arg185 are known as important residues for DNA binding and specificity, but little has been studied for the other residues. Here we show that Gly184 is another F-helix residue critical for the transcriptional activation function of CRP. First, glycine was repeatedly selected at CRP position 184 for its unique ability to provide wild type-level transcriptional activation activity. To dissect the glycine requirement, wild type CRP and mutants G184A, G184F, G184S, and G184Y were purified and theirin vitroDNA-binding activity was measured. G184A and G184F displayed reduced DNA binding, which may explain their low transcriptional activation activity. However, G184S and G184Y displayed apparently normal DNA affinity. Therefore, an additional factor is needed to account for the diminished transcriptional activation function in G184S and G184Y, and the best explanation is perturbations in their interaction with RNA polymerase. The fact that glycine is the smallest amino acid could not fully warrant its suitability, as shown in this study. We hypothesize that Gly184 fulfills the dual functions of DNA binding and RNA polymerase interaction by conferring conformational flexibility to the F-helix.