TRANSCRIPTION OF ALPHA-SPECIFIC GENES IN SACCHAROMYCES-CEREVISIAE - DNA-SEQUENCE REQUIREMENTS FOR ACTIVITY OF THE COREGULATOR ALPHA-1
TRANSCRIPTION OF ALPHA-SPECIFIC GENES IN SACCHAROMYCES-CEREVISIAE - DNA-SEQUENCE REQUIREMENTS FOR ACTIVITY OF THE COREGULATOR ALPHA-1
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DOI：
10.1128/mcb.13.11.6866
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发表时间：
1993-11-01
影响因子：
5.3
通讯作者：
SPRAGUE, GF
中科院分区：
文献类型：
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作者：
HAGEN, DC;BRUHN, L;SPRAGUE, GF
酿酒酵母菌α特异性基因的转录激活是由MCM1和alpha1两种蛋白调控的，这两种蛋白与α特异性基因上游的DNA序列结合，称为P'Q元件。MCM1和alpha1都不能单独有效地结合P'Q元件。然而，它们以一种既需要P'序列（MCM1的弱结合位点）又需要Q序列（被认为是alpha1的结合位点）的方式协同结合。我们分析了STE3基因P'Q元件的点突变集合，以确定单个碱基对对α特异性基因转录的重要性。在10bp的保守Q序列中，只有三个位置的突变强烈影响体内的转录激活。这些相同的突变不影响MCM1单独显示的与P'Q的弱结合。体外DNA结合实验显示，突变序列形成三元P'Q-MCM1-alpha1复合物的能力与体内转录被激活的程度直接相关。因此，α 1和MCM1协同结合P'Q元件的能力对于α特异性基因的激活至关重要。在所有天然α特异性基因中，Q序列与P′的简并侧相邻。为了测试这种几何结构的重要性，我们创建了几个新颖的P、P'和Q序列并置。当Q序列与简并侧相反时，复合QP’元件在体内作为启动子元件无活性，在体外不能形成稳定的三元QP’-MCM1-alpha1复合物。我们还发现，在MCM1强结合位点上添加一个Q序列可以添加α。1到复合体。这一发现，再加上观察到Q元素点突变影响三元复合体的形成，但不影响MCM1的弱结合，支持了Q序列作为alpha1结合位点的观点。
Transcription activation of alpha-specific genes in saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha.1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha1.