McElroy Craig, Ihms Elihu, Yadav Deepak Kumar, Holmquist Melody, Wadwha Vibhuti, Wysocki Vicki, Gollnick Paul, Foster Mark
Ohio State Biochemistry Program.
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210.
bioRxiv. 2023 Jun 30:2023.06.29.547145. doi: 10.1101/2023.06.29.547145.
Cellular production of tryptophan is metabolically expensive and tightly regulated. The small zinc binding Anti-TRAP protein (AT), which is the product of the gene, is upregulated in response to accumulating levels of uncharged tRNA through a T-box antitermination mechanism. AT binds to the undecameric ring-shaped protein TRAP ( RNA Binding Attenuation Protein), thereby preventing it from binding to the leader RNA. This reverses the inhibitory effect of TRAP on transcription and translation of the operon. AT principally adopts two symmetric oligomeric states, a trimer (AT) featuring a three-helix bundle, or a dodecamer (AT) comprising a tetrahedral assembly of trimers, whereas only the trimeric form has been shown to bind and inhibit TRAP. We demonstrate the utility of native mass spectrometry (nMS) and small-angle x-ray scattering (SAXS), together with analytical ultracentrifugation (AUC) for monitoring the pH and concentration-dependent equilibrium between the trimeric and dodecameric structural forms of AT. In addition, we report the use of solution nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AT, while heteronuclear N relaxation measurements on both oligomeric forms of AT provide insights into the dynamic properties of binding-active AT and binding-inactive AT, with implications for TRAP inhibition.
色氨酸的细胞生成在代谢上成本高昂且受到严格调控。小锌结合抗TRAP蛋白（AT）是该基因的产物，它通过T盒抗终止机制响应无电荷tRNA积累水平的升高而上调。AT与十一聚体环状蛋白TRAP（RNA结合衰减蛋白）结合，从而阻止其与前导RNA结合。这逆转了TRAP对操纵子转录和翻译的抑制作用。AT主要呈现两种对称的寡聚状态，一种是具有三螺旋束的三聚体（AT₃），另一种是由三聚体的四面体组装而成的十二聚体（AT₁₂），而只有三聚体形式已被证明能结合并抑制TRAP。我们展示了采用基质辅助激光解吸/电离飞行时间质谱（nMS）和小角X射线散射（SAXS）以及分析超速离心（AUC）来监测AT三聚体和十二聚体结构形式之间pH和浓度依赖性平衡的效用。此外，我们报告了使用溶液核磁共振（NMR）光谱来确定AT的溶液结构，而对AT两种寡聚形式的异核N弛豫测量为结合活性AT和非结合活性AT的动态特性提供了见解，这对TRAP抑制具有重要意义。
