Subramanian Mohan, Qiao Wen-ben, Khanam Nurussaba, Wilkins Olivia, Der Sandy D, Lalich Jonathan D, Bognar Andrew L
Department of Medical Genetics and Microbiology, University of Toronto, Medical Sciences Building Room 4383, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada.
Mol Microbiol. 2005 Jul;57(1):53-69. doi: 10.1111/j.1365-2958.2005.04663.x.
The mechanisms mediating responses to glycine withdrawal in budding yeast were studied using a genome-wide profiling approach. A striking pattern of repressed expression of genes with an enrichment for those involved in one-carbon metabolism and AMP biosynthesis was revealed. Sequence analysis of the promoters for the most severely repressed genes identified a conserved sequence, TGACTC, a known binding site for the transcription factors Gcn4p and Bas1p. Loss of BAS1 abolished or significantly reduced the repression of these genes in response to glycine removal but this phenotype was much less apparent in the absence of BAS2 or GCN4. Addition of a Bas1p-LexA fusion protein to a strain with a LexAop-LacZ fusion showed a strong glycine effect both in a BAS2 and a bas2 background. A Bas1p-VP16 fusion protein activated expression in a bas1bas2 strain but no glycine effect was observed while a Bas1p-Bas2p fusion protein activated expression to a lesser extent with a slight stimulation by glycine. These results suggest that glycine affects Bas1p activation of transcription rather than DNA binding and that Bas2p is not required for this affect. Glycine withdrawal repressed many of the same genes as addition of adenine, a process known to be dependent on Bas1p. However, the glycine response is independent of adenine repression, because glycine regulation occurs normally in ade strains. We did not see any difference in the degree of stimulation by glycine in the presence or absence of adenine even in Ade+ strains. Glycine regulation was also found to be dependent on an intact SHM2 gene, which encodes cytoplasmic serine hydroxymethyltransferase. A reporter plasmid containing a DNA sequence from the GCV2 promoter which confers glycine regulation on heterologous genes was introduced into the yeast deletion set to screen for genes required for glycine regulation. A number of genes, including BAS1 were required for activation by glycine but only the SHM2 gene was required for repression in the absence of glycine. We also showed that regulation of the SHM2 promoter by glycine requires Bas1p but not Bas2p or Gcn4p using a beta-galactosidase reporter. The response of the promoter to glycine required an intact SHM2 gene but was restored in a shm2 strain by addition of formate to the medium.
利用全基因组分析方法研究了出芽酵母中对甘氨酸缺失作出反应的机制。研究揭示了一种显著的基因表达抑制模式，这些基因在参与一碳代谢和AMP生物合成的基因中富集。对抑制最严重的基因的启动子进行序列分析，确定了一个保守序列TGACTC，这是转录因子Gcn4p和Bas1p的已知结合位点。BAS1的缺失消除或显著降低了这些基因对甘氨酸去除的抑制作用，但在没有BAS2或GCN4的情况下，这种表型不太明显。将Bas1p-LexA融合蛋白添加到带有LexAop-LacZ融合的菌株中，在BAS2和bas2背景下均显示出强烈的甘氨酸效应。Bas1p-VP16融合蛋白在bas1bas2菌株中激活表达，但未观察到甘氨酸效应，而Bas1p-Bas2p融合蛋白激活表达的程度较小，且受到甘氨酸的轻微刺激。这些结果表明，甘氨酸影响Bas1p的转录激活而非DNA结合，且这种影响不需要Bas2p。甘氨酸缺失抑制的许多基因与添加腺嘌呤时抑制的基因相同，这一过程已知依赖于Bas1p。然而，甘氨酸反应独立于腺嘌呤抑制，因为甘氨酸调节在ade菌株中正常发生。即使在Ade+菌株中，我们也未观察到在有或没有腺嘌呤的情况下甘氨酸刺激程度的差异。还发现甘氨酸调节依赖于完整的SHM2基因，该基因编码细胞质丝氨酸羟甲基转移酶。将含有来自GCV2启动子的DNA序列（该序列赋予异源基因甘氨酸调节作用）的报告质粒引入酵母缺失文库，以筛选甘氨酸调节所需的基因。许多基因，包括BAS1，是甘氨酸激活所必需的，但在没有甘氨酸的情况下抑制作用仅需要SHM2基因。我们还使用β-半乳糖苷酶报告基因表明，甘氨酸对SHM2启动子的调节需要Bas1p，而不需要Bas2p或Gcn4p。启动子对甘氨酸的反应需要完整的SHM2基因，但在shm2菌株中通过向培养基中添加甲酸可恢复这种反应。
