Identifying DNA sequences recognized by a transcription factor using a bacterial one-hybrid system
Identifying DNA sequences recognized by a transcription factor using a bacterial one-hybrid system
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DOI：
10.1038/nprot.2006.6
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Wolfe, Scot A.
中科院分区：
文献类型：
--
作者：
Meng, Xiangdong;Wolfe, Scot A.
基于细菌的相互作用捕获系统为识别相互作用的大分子提供了一种强有力的方法。当在遗传选择的背景下进行时，相互作用的对可以从大的组合文库中快速分离出来。这项技术已被应用于从大型随机文库中鉴定转录因子(Tf)的DNA结合序列。该方法使用共顺反子HIS3-URA3报告盒上游的随机结合位点文库。URA3报告器允许通过反选择将自激活序列从文库中移除。HIS3报告器允许从文库中分离出被转录因子识别的序列，其中转录激活是通过将转录因子与RNA聚合酶的阿尔法亚单位融合而介导的。这项技术可以用来表征单体、同二聚体和异二聚体的DNA结合域，一旦构建了合适的文库，结合部位就可以在大约10天内确定。细菌单杂交系统允许搜索比相应的酵母单杂交系统更大的文库，并且与SELEX不同，它不需要纯化转铁蛋白(S)。然而，使用细菌系统可以搜索的结合位点文库的复杂性比SELEX更有限，并且一些真核因子可能不能在细菌系统中有效地表达或折叠。
Bacterial-based interaction trap systems provide a powerful method to identify interacting macromolecules. When carried out in the context of a genetic selection, interacting pairs can be rapidly isolated from large combinatorial libraries. This technology has been adapted to allow the identification of DNA-binding sequences for a transcription factor (TF) from a large randomized library. This procedure uses a library of randomized binding sites upstream of a cocistronic HIS3-URA3 reporter cassette. The URA3 reporter allows self-activating sequences to be removed from the library through counter-selection. The HIS3 reporter allows sequences that are recognized by a TF to be isolated from the library, where transcriptional activation is mediated by fusion of the TF to the alpha-subunit of RNA polymerase. This technology can be used to characterize monomeric, homodimeric and heterodimeric DNA-binding domains and, once a suitable library is constructed, binding sites can be identified in approximately 10 d. The bacterial one-hybrid system allows larger libraries to be searched than the corresponding yeast one-hybrid system and, unlike SELEX, it does not require purification of the TF(s). The complexity of the binding site libraries that can be searched using the bacterial system is, however, more limited than SELEX, and some eukaryotic factors may not express or fold efficiently in the bacterial system.