Combinatorial targeting of ribbon-helix-helix artificial transcription factors to chimeric recognition sites.
Combinatorial targeting of ribbon-helix-helix artificial transcription factors to chimeric recognition sites.
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DOI：
10.1093/nar/gks314
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发表时间：
2012-08
影响因子：
14.9
通讯作者：
Hayes F
中科院分区：
文献类型：
--
作者：
Zampini M;Hayes F
人工转录因子（ATFs）是调节内源基因表达和精确基因组编辑的有效合成生物学工具。转录因子的带-螺旋-螺旋（ribbon-helix-helix，RHH）超家族广泛存在于细菌和古细菌中。该家族中的主要DNA结合决定簇包括双链反平行β-折叠（条带），其中一对8残基序插入到大沟中。在这里，我们证明了不同的RHH蛋白质的条带是紧凑和便携的元素，可以移植到一个共同的α-螺旋支架产生活性ATF。杂合蛋白协同识别具有核心四聚体盒的DNA位点，其功能间隔由α-螺旋骨架之间的相互作用决定。这些相互作用还促进具有不同移植条带但相同主链的嵌合体与在体外或体内承载每种蛋白质的同源盒的合成位点的组合结合。与涉及单个蛋白质结合的ATF相比，相互作用的杂合蛋白质的复合组装提供了与DNA识别的组合方法相关的潜在优势。此外，新类别的RHH ATF可用于重新设计转录回路，或者可以用亲和标签、荧光部分或用于细菌和古细菌中的靶向基因组标记和操作的其他元件来增强。
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.
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