HMG盒
DNA结合位点
DNA结合域
绑定域
DNA
生物
蛋白质-DNA相互作用
单链结合蛋白
B3域
基因
DNA钳
结合位点
体外重组
分子生物学
DNA结合蛋白
遗传学
发起人
基因表达
转录因子
分子克隆
逆转录酶
核糖核酸
作者
Jingdong Liu,Thomas E. Wilson,Jeffrey Milbrandt,Mark Johnston
出处
期刊:Methods
[Elsevier]
日期:1993-08-01
卷期号:5 (2): 125-137
被引量:47
标识
DOI:10.1006/meth.1993.1017
摘要
We describe genetic methods using yeast to analyze a DNA-binding protein to determine (i) the sequence of the DNA sites to which the protein binds and (ii) the location of the domain and specific amino acid residues in the protein responsible for DNA binding. These methods take advantage of the fact that a hybrid protein consisting of a particular DNA-binding domain and a transcriptional activation domain activates expression of a reporter gene that contains binding sites for the DNA-binding domain. We describe two applications of these methods. First, DNA fragments that contain binding sites for the DNA-binding protein of interest can be recovered from a library of fragments by their ability to mediate transcriptional activation of a reporter gene. If enough DNA fragments are identified, the consensus sequence of the DNA-binding site can usually be recognized. In addition, some of the DNA fragments may be derived from actual target genes regulated by the DNA-binding protein, and therefore these fragments might be used to identify such target genes. Second, a reporter gene whose expression inhibits cell growth and whose promoter contains binding sites for the DNA-binding protein can be used to select mutants defective in the DNA-binding domain. This procedure allows one to localize the DNA-binding domain within the protein and to identify amino acids important for DNA binding. The mutations that inactivate the DNA-binding domain are highly informative, since the method avoids the recovery of "uninteresting" mutations that simply destabilize the protein or prevent its synthesis. In principle, the methods we describe can be applied to any DNA-binding protein.
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