Ebbole D J, Paluh J L, Plamann M, Sachs M S, Yanofsky C
Department of Biological Sciences, Stanford University, California 94305.
Mol Cell Biol. 1991 Feb;11(2):928-34. doi: 10.1128/mcb.11.2.928-934.1991.
CPCI, the principal regulatory protein required for cross-pathway control of amino acid biosynthetic genes in Neurospora crassa, contains a domain similar to the DNA-binding domain of GCN4, the corresponding general regulator in Saccharomyces cerevisiae. We examined binding by CPC1 synthesized in vitro and by CPC1 present in N. crassa whole-cell extracts. CPCI from both sources was shown to bind to the DNA sequence 5'-ATGACTCAT-3', which is also the preferred recognition sequence of GCN4, CPC1 was confirmed as the source of DNA-binding activity in extracts by immunoblotting. Slightly mobility differences between DNA complexes containing CPCI synthesized in vitro and CPC1 in mycelial extracts were observed. Analyses of N. crassa extracts from different stages of asexual development revealed that CPC1 was abundant immediately following spore germination and through early mycelial growth but was scarce subsequently. CPC1 levels could be increased at any time by imposing amino acid starvation. Copies of the CPC1 response element are located upstream of several genes regulated by cross-pathway control, including cpc-1 itself.
CPCI是粗糙脉孢菌中氨基酸生物合成基因交叉途径控制所需的主要调节蛋白，它含有一个与酿酒酵母中相应的通用调节因子GCN4的DNA结合结构域相似的结构域。我们检测了体外合成的CPC1以及粗糙脉孢菌全细胞提取物中存在的CPC1的结合情况。来自这两种来源的CPCI都被证明能与DNA序列5'-ATGACTCAT-3'结合，该序列也是GCN4的首选识别序列。通过免疫印迹法证实CPC1是提取物中DNA结合活性的来源。观察到含有体外合成的CPCI的DNA复合物与菌丝体提取物中的CPC1之间存在轻微的迁移率差异。对粗糙脉孢菌无性发育不同阶段的提取物分析表明，CPC1在孢子萌发后紧接着以及早期菌丝生长阶段含量丰富，但随后含量稀少。通过施加氨基酸饥饿，CPC1水平可在任何时候升高。CPC1反应元件的拷贝位于几个受交叉途径控制调节的基因的上游，包括cpc-1自身。
