Halvorsen Y C, Nandabalan K, Dickson R C
Department of Biochemistry, University of Kentucky, Lexington 40536.
J Biol Chem. 1990 Aug 5;265(22):13283-9.
The LAC9 protein of Kluyveromyces lactis activates transcription by binding to upstream activating sequences lying in front of genes of the lactose-galactose regulon. LAC9 belongs to a family of fungal proteins having a conserved domain containing 6 cysteines. This domain, termed a C6 zinc finger, is thought to bind one zinc atom and to play a vital role in DNA binding. To further characterize the DNA-binding domain of LAC9, we have developed a procedure to produce and to purify milligram amounts of LAC9 peptides. The two larger peptides, one containing amino acids 1-228 and the other containing amino acids 85-228, formed dimers in solution and bound DNA specifically as a dimer. The smallest LAC9 peptide, amino acids 85-160, failed to dimerize and did not bind DNA. Atomic absorption spectroscopy revealed that each LAC9 monomer coordinated two zinc atoms, not one, as had been predicted. This result suggests, as does previously published data, that the C6 zinc finger domain has a unique conformation that may represent a new type of DNA-binding motif.
乳酸克鲁维酵母的LAC9蛋白通过与乳糖-半乳糖操纵子基因前的上游激活序列结合来激活转录。LAC9属于一类具有保守结构域的真菌蛋白家族，该结构域含有6个半胱氨酸。这个结构域被称为C6锌指，被认为能结合一个锌原子，并在DNA结合中起关键作用。为了进一步表征LAC9的DNA结合结构域，我们开发了一种生产和纯化毫克量LAC9肽的方法。两种较大的肽，一种包含1-228个氨基酸，另一种包含85-228个氨基酸，在溶液中形成二聚体，并作为二聚体特异性结合DNA。最小的LAC9肽，85-160个氨基酸，未能形成二聚体，也不结合DNA。原子吸收光谱显示，每个LAC9单体配位两个锌原子，而不是如先前预测的一个锌原子。这一结果与先前发表的数据一样表明，C6锌指结构域具有独特的构象，可能代表一种新型的DNA结合基序。
