Genetic regulation mediated by thiamin pyrophosphate-binding motif in Saccharomyces cerevisiae
Genetic regulation mediated by thiamin pyrophosphate-binding motif in Saccharomyces cerevisiae
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DOI：
10.1111/j.1365-2958.2005.04835.x
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发表时间：
2005-10-01
影响因子：
3.6
通讯作者：
Akaji, K
中科院分区：
文献类型：
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作者：
Nosaka, K;Onozuka, M;Akaji, K
编码参与硫胺素代谢的酶的酿酒酵母基因（THI基因）的表达被外源硫胺素协同抑制，并在缺乏硫胺素的情况下被诱导。在该酵母THI调节系统中，THI调节系统主要在转录水平起作用，焦磷酸硫胺素（TDP）似乎充当辅阻遏物，并且遗传研究已经鉴定了三种正调节因子（Thi2p、Thi3p和Pdc2p）。我们在DNA微阵列分析中发现，THI基因的表达增加了10至90倍，以响应硫胺素剥夺，同样，THI2和THI3的表达分别增加了17倍和3倍。从阻遏转移到诱导培养基后，THI2和THI3的启动子活性与THI基因之一的PHO3的活性平行增加。THI3启动子活性的刺激通过THI3的缺失而减弱，指示THI3的自动调节。当THI2在thi3 Delta菌株中由酵母GAL 1启动子表达时，或当THI3在thi2 Delta菌株中表达时，THI基因未被诱导，表明Thi2p和Thi3p同时参与诱导。当在thi3 Delta菌株中产生缺乏TDP结合活性的突变体Thi3p蛋白时，即使在硫胺素充足的条件下也表达THI基因。这一结果支持了Thi3p感知THI调节系统的细胞内信号以施加转录控制的假设。此外，证明了Thi2p和Thi3p相互结合，并且这种相互作用被外源性硫胺素部分减弱，这表明Thi2p和Thi3p刺激作为复合物的表达，其功能被与Thi3p结合的TDP干扰。我们讨论的可能性，THI基因的诱导是由激活的复合物引起的细胞内TDP的减少和升高的复合物中的自动调节方式进一步上调THI基因。这是第一次报告的TDP结合基序参与遗传调控。
The expression of genes of Saccharomyces cerevisiae encoding the enzymes involved in the metabolism of thiamin (THI genes) is co-ordinately repressed by exogenous thiamin and induced in the absence of thiamin. In this yeast THI regulatory system acts mainly at the transcriptional level, thiamin pyrophosphate (TDP) seems to serve as a corepressor, and genetic studies have identified three positive regulatory factors (Thi2p, Thi3p and Pdc2p). We found in a DNA microarray analysis that the expression of THI genes increased 10- to 90-fold in response to thiamin deprivation, and likewise, the expression of THI2 and THI3 increased 17-fold and threefold, respectively. After transfer from repressing to inducing medium, the promoter activity of both THI2 and THI3 increased in parallel with that of PHO3, one of THI genes. The stimulation of THI3 promoter activity was diminished by deletion of THI3, indicative of the autoregulation of THI3. The THI genes were not induced when THI2 was expressed from the yeast GAL1 promoter in a thi3 Delta strain or when THI3 was expressed in a thi2 Delta strain, suggesting that Thi2p and Thi3p participate simultaneously in the induction. When mutant Thi3p proteins lacking TDP-binding activity were produced in the thi3 Delta strain, THI genes were expressed even under thiamin-replete conditions. This result supports the hypothesis that Thi3p senses the intracellular signal of the THI regulatory system to exert transcriptional control. Furthermore, Thi2p and Thi3p were demonstrated to bind each other and this interaction was partially diminished by exogenous thiamin, suggesting that Thi2p and Thi3p stimulate the expression as a complex whose function is disturbed by TDP bound to Thi3p. We discuss the possibility that the induction of THI genes is triggered by the activation of the complex attributed to decrease in intracellular TDP and the elevated complex in the autoregulatory fashion further upregulates THI genes. This is the first report of the involvement of the TDP-binding motif in genetic regulation.