Svetlov V, Cooper T G
Department of Microbiology and Immunology, University of Tennessee, Memphis 38163, USA.
J Bacteriol. 1997 Dec;179(24):7644-52. doi: 10.1128/jb.179.24.7644-7652.1997.
Regulated nitrogen catabolic gene transcription in Saccharomyces cerevisiae is mediated by four positive (Gln3p and Gat1p/Nil1p) and negative (Dal80p/Uga43p and Deh1p/Nil2p/GZF3p) regulators which function in opposition to one another. All four proteins contain GATA-type zinc finger domains, and three of them (Gln3p, Dal80p, and Deh1p) have been shown to bind to GATA sequences situated upstream of genes whose expression is sensitive to nitrogen catabolite repression (NCR). The positive regulators, Gln3p and Gat1p, are able to support transcriptional activation when tethered by LexAp to the promoter of a reporter gene whose upstream activation sequences have been replaced with one or more lexA operator sites. Existing data suggest that these four proteins regulate transcription by competing with one another for binding to the GATA sequences which mediate NCR-sensitive gene expression. We show that the minimal Gln3p domain mediating transcriptional activation consists of 13 amino acids with a predicted propensity to form an alpha-helix. Genetic analysis of this region (Gln3p residues 126 to 138, QQNGEIAQLWDFN) demonstrated that alanine may be substituted for the aromatic and acidic amino acids without destroying transcriptional activation potential. Similar substitution of alanine for the two hydrophobic amino acids, isoleucine and leucine, however, destroys activation, as does introduction of basic amino acids in place of the acidic residues or introduction of proline into the center of the sequence. A point mutation in the Gln3p activation region destroys its in vivo ability to support NCR-sensitive DAL5 expression. We find no convincing evidence that NCR regulates Gln3p function by modulating the functioning of its activation region.
酿酒酵母中受调控的氮分解代谢基因转录由四个正向（Gln3p和Gat1p/Nil1p）和负向（Dal80p/Uga43p和Deh1p/Nil2p/GZF3p）调节因子介导，它们相互拮抗发挥作用。所有这四种蛋白质都含有GATA型锌指结构域，其中三种（Gln3p、Dal80p和Deh1p）已被证明能与位于对氮分解代谢物阻遏（NCR）敏感的基因上游的GATA序列结合。正向调节因子Gln3p和Gat1p，当通过LexAp与报告基因的启动子相连时，能够支持转录激活，该报告基因的上游激活序列已被一个或多个lexA操纵位点取代。现有数据表明，这四种蛋白质通过相互竞争结合介导NCR敏感基因表达的GATA序列来调节转录。我们表明，介导转录激活的最小Gln3p结构域由13个氨基酸组成，预测有形成α螺旋的倾向。对该区域（Gln3p的126至138位残基，QQNGEIAQLWDFN）的遗传分析表明，丙氨酸可以取代芳香族和酸性氨基酸而不破坏转录激活潜能。然而，用丙氨酸类似地取代两个疏水氨基酸异亮氨酸和亮氨酸会破坏激活，用碱性氨基酸取代酸性残基或在序列中心引入脯氨酸也会如此。Gln3p激活区域的一个点突变破坏了其在体内支持NCR敏感的DAL5表达的能力。我们没有发现令人信服的证据表明NCR通过调节其激活区域的功能来调节Gln3p的功能。
