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首页> 外文期刊>Journal of Molecular Biology >Isolation of mutations that disrupt cooperative DNA binding by theDrosophila Bicoid protein
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Isolation of mutations that disrupt cooperative DNA binding by theDrosophila Bicoid protein

机译：分离破坏果蝇Bicoid蛋白协同DNA结合的突变

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Cooperative DNA binding is thought to contribute to the ability of the Drosophila melanogaster protein, Bicoid, to stimulate transcription of target genes in precise sub-domains within the embryo. As a first step toward testing this idea, we devised a genetic screen to isolate mutations in Bicoid that specifically disrupt cooperative interactions, but do not disrupt DNA recognition or transcription activation. The screen was carried out in Saccharomyces cerevisiae and 12 cooperativity mutants were identified. The mutations map across most of the Bicoid protein, with some located within the DNA-binding domain (homeodomain). Four homeodomain mutants were characterized in yeast and shown to activate a single-site reporter gene to levels comparable to that of wild-type, indicating that DNA binding per se is not affected. However, these mutants failed to show cooperative coupling between high and low-affinity sites, and showed reduced activation of a reporter gene carrying a natural Drosophila enhancer. Homology modeling indicated that none of the four mutations is in residues that contact DNA. Instead, these residues are likely to interact with other DNA-bound Bicoid monomers or other parts of the Bicoid protein. In vitro, the isolated homeodomains did not show strong cooperativity defects, supporting the idea that other regions of Bicoid are also important for cooperativity. This study describes the first systematic screen to identify cooperativity mutations in a eukaryotic DNA-binding protein.
机译：人们认为合作的DNA结合有助于果蝇果蝇Bicoid刺激胚胎内精确子域中靶基因的转录。作为检验该想法的第一步，我们设计了一种遗传筛选方法，以分离比可oid中的突变，该突变可特异性破坏协同相互作用，但不会破坏DNA识别或转录激活。筛选是在酿酒酵母中进行的，并鉴定了12个协同突变体。突变分布在大多数Bicoid蛋白上，有些位于DNA结合域（同源域）内。在酵母中鉴定了四个同源域突变体，并显示它们可将单点报道基因激活至与野生型相当的水平，表明DNA结合本身不受影响。但是，这些突变体未能显示出高亲和力位点和低亲和力位点之间的协同偶联，并且显示出携带天然果蝇增强子的报告基因的激活减少。同源性建模表明，这四个突变都不在与DNA接触的残基中。而是，这些残基可能与其他DNA结合的Bicoid单体或Bicoid蛋白的其他部分相互作用。在体外，分离的同源域没有显示出强烈的协同缺陷，这支持了双环类化合物的其他区域对于协同作用也很重要的观点。这项研究描述了第一个系统的筛选，以鉴定真核DNA结合蛋白中的协同突变。

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