Faculty of Life Sciences and Manchester Interdisciplinary Biocentre, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
Nucleic Acids Res. 2012 Aug;40(14):6673-82. doi: 10.1093/nar/gks314. Epub 2012 Apr 9.
Artificial transcription factors (ATFs) are potent synthetic biology tools for modulating endogenous gene expression and precision genome editing. The ribbon-helix-helix (RHH) superfamily of transcription factors are widespread in bacteria and archaea. The principal DNA binding determinant in this family comprises a two-stranded antiparallel β-sheet (ribbons) in which a pair of eight-residue motifs insert into the major groove. Here, we demonstrate that ribbons of divergent RHH proteins are compact and portable elements that can be grafted into a common α-helical scaffold producing active ATFs. Hybrid proteins cooperatively recognize DNA sites possessing core tetramer boxes whose functional spacing is dictated by interactions between the α-helical backbones. These interactions also promote combinatorial binding of chimeras with different transplanted ribbons, but identical backbones, to synthetic sites bearing cognate boxes for each protein either in vitro or in vivo. The composite assembly of interacting hybrid proteins offers potential advantages associated with combinatorial approaches to DNA recognition compared with ATFs that involve binding of a single protein. Moreover, the new class of RHH ATFs may be utilized to re-engineer transcriptional circuits, or may be enhanced with affinity tags, fluorescent moieties or other elements for targeted genome marking and manipulation in bacteria and archaea.
人工转录因子（ATFs）是一种强大的合成生物学工具，可用于调节内源性基因表达和精确的基因组编辑。转录因子的螺旋-环-螺旋（RHH）超家族在细菌和古菌中广泛存在。该家族中的主要 DNA 结合决定因素包括一个由两条反平行 β 链组成的双股螺旋（ribbons），其中一对由 8 个残基组成的motif 插入到主沟中。在这里，我们证明了不同的 RHH 蛋白的 ribbon 是紧凑且可移植的元件，可以嫁接到一个常见的 α-螺旋支架上，产生有活性的 ATFs。杂交蛋白协同识别具有核心四聚体盒的 DNA 位点，其功能间距由α-螺旋骨架之间的相互作用决定。这些相互作用还促进了具有不同移植 ribbon 的嵌合体与具有每个蛋白的同源盒的合成位点的组合结合，无论是在体外还是体内。相互作用的杂交蛋白的复合组装提供了与涉及单个蛋白结合的 ATFs 相比，与 DNA 识别相关的组合方法的潜在优势。此外，新型 RHH ATFs 可用于重新设计转录电路，或者可以通过亲和标签、荧光部分或其他元件进行增强，以用于细菌和古菌中的靶向基因组标记和操作。
