Bai Y L, Kohlhaw G B
Department of Biochemistry, Purdue University, West Lafayette, IN 47907.
Nucleic Acids Res. 1991 Nov 11;19(21):5991-7. doi: 10.1093/nar/19.21.5991.
The transcriptional activator LEU3 of Saccharomyces cerevisiae belongs to a family of lower eukaryotic DNA binding proteins with a well-conserved DNA binding motif known as the Zn(II)2Cys6 binuclear cluster. We have constructed mutations in LEU3 that affect either one of the conserved cysteines (Cys47) or one of several amino acids located within a variable subregion of the DNA binding motif. LEU3 proteins with a mutation at Cys47 were very poor activators which could not be rescued by supplying Zn(II) to the growth medium. Mutations within the variable subregion were generally well-tolerated. Only two of seven mutations in this region generated poor activators, and both could be reactivated by Zn(II) supplements. Three of the other five mutations gave rise to activators that were better than wild type. One of these, His50Cys, exhibited a 1.5 fold increase in in vivo target gene activation and a notable increase in the affinity for target DNA. The properties of the His50Cys mutant are discussed in terms of a variant structure of the DNA binding motif. During the course of this work, evidence was obtained suggesting that only one of the two LEU3 protein-DNA complexes routinely seen actually activates transcription. The other (which may contain an additional protein factor) does not.
酿酒酵母的转录激活因子LEU3属于一类低等真核生物DNA结合蛋白家族，其具有一个保守性良好的DNA结合基序，即Zn(II)2Cys6双核簇。我们构建了LEU3中的突变，这些突变影响保守半胱氨酸之一（Cys47）或位于DNA结合基序可变亚区内的几个氨基酸之一。在Cys47处发生突变的LEU3蛋白是非常差的激活因子，向生长培养基中供应Zn(II)也无法挽救它们。可变亚区内的突变通常耐受性良好。该区域的七个突变中只有两个产生了较差的激活因子，并且两者都可以通过补充Zn(II)重新激活。其他五个突变中的三个产生了比野生型更好的激活因子。其中之一，His50Cys，在体内靶基因激活方面表现出1.5倍的增加，并且对靶DNA的亲和力显著增加。根据DNA结合基序的变体结构讨论了His50Cys突变体的特性。在这项工作过程中，获得的证据表明，通常看到的两种LEU3蛋白-DNA复合物中只有一种实际上激活转录。另一种（可能包含额外的蛋白质因子）则不激活。
