Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (10): 54-65.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1384
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CHEN Chen(), HUANG Zhi-yang, YU Hai-yan, YUAN Hai-bin, TIAN Huai-xiang()
Received:
2021-11-03
Online:
2022-10-26
Published:
2022-11-11
Contact:
TIAN Huai-xiang
E-mail:chenchen@sit.edu.cn;tianhx@sit.edu.cn
CHEN Chen, HUANG Zhi-yang, YU Hai-yan, YUAN Hai-bin, TIAN Huai-xiang. Research Technology and Progress in Transcriptional Regulation in Prokaryotes[J]. Biotechnology Bulletin, 2022, 38(10): 54-65.
研究技术 Method | 英文名称/缩写 Name/Abbreviation | 优缺点 Advantages/Disadvantages | 适用性 Applicability | 参考文献 Reference | |
---|---|---|---|---|---|
体外方法 | 凝胶电泳迁移率实验 | EMSA | 方法简单、灵敏度高,放射性标记探针安全性低成本高,电泳运行条件下蛋白质-DNA复合体不稳定 | 适用于验证转录因子与假定DNA结合位点直接相互作用及结合位点突变对结合作用的影响 | [ |
等温滴定量热法 | ITC | 蛋白质无需固定化或修饰,样品消耗量少,可区分结合常数相近的配体相互作用及比较结构与结合作用的关系,对温度适应范围广但难以解释复杂系统中的相互作用 | 适用于成分简单的超高/超低亲和力相互作用系统及复杂的相互作用,可获得丰富的热力学信息 | [ | |
DNase I footprinting技术 | DNase I footprinting | 分辨率高、可区分同一DNA片段多个不连续结合位点,但需要较多蛋白质才能产生清晰足迹,易产生超敏位点受到切割 | 适用于未纯化蛋白样品的检测,判断同一片段是否存在多个结合位点获得结合序列及比较各自亲和力 | [ | |
微量热泳动技术 | MST | 对相互作用的分子大小或质量无选择性,具有较好的适用性,对结合亲和力准确测定,可检测低至pM级别的结合亲和力,可在溶液环境中进行,无需固定分子避免结合假阳性 | 适用于结合亲和力弱,样品量小,样品所处环境复杂的情况 | [ | |
体内方法 | 染色质-免疫共沉淀技术 | ChIP | 接近细胞内真实情况,可研究转录因子对启动子结合的动态过程,但实验重复性不佳,获得良好实验结果对经验依赖性较高,对实验环境的要求严格 | 适用于确定转录因子修饰位置及低丰度转录因子结合分析 | [ |
细菌单杂交 | B1H | 细菌转化效率高构建文库质粒容量更大,无需复杂的仪器,转录因子需要能在大肠杆菌中表达,可能存在假阳性和假阴性的情况,结果需要进一步验证 | 适用于未纯化蛋白样品的检测,缺少相应仪器,用分子生物学手段进行筛选,转录因子要求能在大肠杆菌中表达,可发现新的转录因子 | [ |
Table 1 Summary of research techniques on transcriptional regulation in prokaryotes
研究技术 Method | 英文名称/缩写 Name/Abbreviation | 优缺点 Advantages/Disadvantages | 适用性 Applicability | 参考文献 Reference | |
---|---|---|---|---|---|
体外方法 | 凝胶电泳迁移率实验 | EMSA | 方法简单、灵敏度高,放射性标记探针安全性低成本高,电泳运行条件下蛋白质-DNA复合体不稳定 | 适用于验证转录因子与假定DNA结合位点直接相互作用及结合位点突变对结合作用的影响 | [ |
等温滴定量热法 | ITC | 蛋白质无需固定化或修饰,样品消耗量少,可区分结合常数相近的配体相互作用及比较结构与结合作用的关系,对温度适应范围广但难以解释复杂系统中的相互作用 | 适用于成分简单的超高/超低亲和力相互作用系统及复杂的相互作用,可获得丰富的热力学信息 | [ | |
DNase I footprinting技术 | DNase I footprinting | 分辨率高、可区分同一DNA片段多个不连续结合位点,但需要较多蛋白质才能产生清晰足迹,易产生超敏位点受到切割 | 适用于未纯化蛋白样品的检测,判断同一片段是否存在多个结合位点获得结合序列及比较各自亲和力 | [ | |
微量热泳动技术 | MST | 对相互作用的分子大小或质量无选择性,具有较好的适用性,对结合亲和力准确测定,可检测低至pM级别的结合亲和力,可在溶液环境中进行,无需固定分子避免结合假阳性 | 适用于结合亲和力弱,样品量小,样品所处环境复杂的情况 | [ | |
体内方法 | 染色质-免疫共沉淀技术 | ChIP | 接近细胞内真实情况,可研究转录因子对启动子结合的动态过程,但实验重复性不佳,获得良好实验结果对经验依赖性较高,对实验环境的要求严格 | 适用于确定转录因子修饰位置及低丰度转录因子结合分析 | [ |
细菌单杂交 | B1H | 细菌转化效率高构建文库质粒容量更大,无需复杂的仪器,转录因子需要能在大肠杆菌中表达,可能存在假阳性和假阴性的情况,结果需要进一步验证 | 适用于未纯化蛋白样品的检测,缺少相应仪器,用分子生物学手段进行筛选,转录因子要求能在大肠杆菌中表达,可发现新的转录因子 | [ |
Fig. 1 Basic principle of EMSA experiment Asterisks indicate labeled probes,TFs indicate transcription factors,and TFBS indicate transcription factor binding sites
Fig. 2 Basic principle of DNase I footprint technology Adding appropriate concentration of deoxyribonuclease I(DNase I)to label strand in the DNA fragment,the DNA motif bound to the target protein will not be hydrolyzed by DNase I. After comparing the autoradiograph of the DNA fragment not bound to the target protein,interrupted DNA gradient bands can be obtained
Fig. 3 Basic principle of ChIP experiment Cross-link the target protein with DNA in vivo through a cross-linking agent,and then fragment the DNA and protein complex. After that,precipitate with the specific antibody of the target protein to enrich the DNA fragments bound to the target protein. Reversal of crosslink and DNA extraction,finally analysis of purified DNA
Fig. 4 Basic principle of MST experiment A:Fluorescence detector. B:MST optical element schematic diagram. C:MST experiment signal curve. D:MST combination experiment
Fig. 5 Basic principle of ITC experiment The titration of reactants of known concentration into the sample causes the reaction between the components in the sample cell to be endothermic or exothermic. The temperature compensation system maintains a constant temperature difference between the sample cell and the reference cell,and the data are recorded to simulate the integrated heating isotherm. After that,information about the reaction enthalpy change,binding affinity and binding stoichiometry can be obtained.
Fig. 6 Basic principle of bacterial one hybrid system A:Transcription factor recognition binding sites activate the expressions of the reporter genes. B:Construct α-TF expression vector and binding site library respectively. C:Eliminate self-activation to purify binding site library. D:Selection and analysis of binding sites
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