To create bacterial transcription “circuits” for biotechnology, one approach is to recombine natural transcription factors, promoters, and operators. Additional novel functions can be engineered from existing transcription factors such as the E. coli AraC transcriptional activator, for which binding to DNA is modulated by binding L-arabinose. Here, we engineered chimeric AraC/XylS transcription activators that recognized ara DNA binding sites and responded to varied effector ligands. The first step, identifying domain boundaries in the natural homologs, was challenging because (i) no full-length, dimeric structures were available and (ii) extremely low sequence identities (≤10%) among homologs precluded traditional assemblies of sequence alignments. Thus, to identify domains, we built and aligned structural models of the natural proteins. The designed chimeric activators were assessed for function, which was then further improved by random mutagenesis. Several mutational variants were identified for an XylS•AraC chimera that responded to benzoate; two enhanced activation to near that of wild-type AraC. For an RhaR•AraC chimera, a variant with five additional substitutions enabled transcriptional activation in response to rhamnose. These five changes were dispersed across the protein structure, and combinatorial experiments testing subsets of substitutions showed significant non-additivity. Combined, the structure modeling and epistasis suggest that the common AraC/XylS structural scaffold is highly interconnected, with complex intra-protein and inter-domain communication pathways enabling allosteric regulation. At the same time, the observed epistasis and the low sequence identities of the natural homologs suggest that the structural scaffold and function of transcriptional regulation are nevertheless highly accommodating of amino acid changes.

中文翻译：

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AraC/XylS 同系物高度互连的结构支架内的变构调节可耐受多种氨基酸变化

要为生物技术创建细菌转录“回路”，一种方法是重组天然转录因子、启动子和操纵子。可以从现有的转录因子（如大肠杆菌AraC 转录激活因子）设计其他新功能，通过结合 L-阿拉伯糖来调节与 DNA 的结合。在这里，我们设计了识别ara的嵌合 AraC/XylS 转录激活剂DNA 结合位点并对不同的效应配体作出反应。第一步，识别天然同源物中的域边界，具有挑战性，因为 (i) 没有可用的全长二聚体结构，以及 (ii) 同源物中极低的序列同一性 (≤10%) 排除了传统的序列比对组装。因此，为了识别域，我们构建并对齐了天然蛋白质的结构模型。对设计的嵌合激活剂进行功能评估，然后通过随机诱变进一步改进。对苯甲酸盐有反应的 XylS•AraC 嵌合体鉴定了几种突变变体；两个增强的激活接近野生型 AraC。对于 RhaR•AraC 嵌合体，具有五个额外替换的变体启用转录激活以响应鼠李糖。这五个变化分散在整个蛋白质结构中，测试替代子集的组合实验显示出显着的非可加性。结合结构建模和上位性表明，常见的 AraC/XylS 结构支架是高度互连的，具有复杂的蛋白质内和域间通讯途径，可实现变构调节。同时，观察到的上位性和天然同源物的低序列同一性表明结构支架和转录调节功能仍然高度适应氨基酸变化。结构建模和上位性表明，常见的 AraC/XylS 结构支架是高度互连的，具有复杂的蛋白质内和域间通讯途径，可实现变构调节。同时，观察到的上位性和天然同源物的低序列同一性表明结构支架和转录调节功能仍然高度适应氨基酸变化。结构建模和上位性表明，常见的 AraC/XylS 结构支架是高度互连的，具有复杂的蛋白质内和域间通讯途径，可实现变构调节。同时，观察到的上位性和天然同源物的低序列同一性表明结构支架和转录调节功能仍然高度适应氨基酸变化。