Strauss J, Mach R L, Zeilinger S, Hartler G, Stöffler G, Wolschek M, Kubicek C P
Abteilung für Mikrobielle Biochemie, Institut für Biochemische Technologie und Mikrobiologie, Wien, Austria.
FEBS Lett. 1995 Nov 27;376(1-2):103-7. doi: 10.1016/0014-5793(95)01255-5.
In order to investigate the mechanism of carbon catabolite repression in the industrially important fungus Trichoderma reesei, degenerated PCR-primers were designed to amplify a 0.7-bp fragment of the cre1 gene, which was used to clone the entire gene. It encodes a 402-amino acid protein with a calculated M(r) of 43.6 kDa. Its aa-sequence shows 55.6% and 54.7% overall similarity to the corresponding genes of Aspergillus nidulans and A. niger, respectively. Similarity was restricted to the aa-region containing the C2H2 zinc finger and several aa-regions rich in proline and basic amino acids, which may be involved in the interaction with other proteins. Another aa-region rich in the SPXX-motif that has been considered analogous to a region of yeast RGR1p, was instead identified as a domain occurring in several eucaryotic transcription factors. The presence of the cre1 translation product was demonstrated with polyclonal antibodies against Cre1, which identified a protein of 43 (+/- 2) kDa in cell-free extracts from T. reesei. A Cre1 protein fragment from the two zinc fingers to the region similar to the aa-sequence of eucaryotic transcription factors, was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. EMSA and in vitro footprinting revealed binding of the fusion protein to the sequence 5'-GCGGAG-3', which matches well with the A. nidulans consensus sequence for CreA binding (5'-SYGGRG-3'). Cell-free extracts of T. reesei formed different complexes with DNA-fragments carrying this binding sites, and the presence of Cre1 and additional proteins in these complexes was demonstrated. We conclude that T. reesei Cre1 is the functional homologue of Aspergillus CreA and that it binds to its target sequence probably as a protein complex.
为了研究工业重要真菌里氏木霉中碳分解代谢物阻遏的机制，设计了简并PCR引物来扩增cre1基因的一个0.7kb片段，该片段用于克隆整个基因。它编码一个402个氨基酸的蛋白质，计算分子量为43.6 kDa。其氨基酸序列与构巢曲霉和黑曲霉的相应基因分别显示出55.6%和54.7%的总体相似性。相似性局限于含有C2H2锌指的氨基酸区域以及几个富含脯氨酸和碱性氨基酸的区域，这些区域可能参与与其他蛋白质的相互作用。另一个富含SPXX基序的氨基酸区域，该区域被认为类似于酵母RGR1p的一个区域，相反被鉴定为存在于几种真核转录因子中的一个结构域。用抗Cre1的多克隆抗体证明了cre1翻译产物的存在，该抗体在里氏木霉的无细胞提取物中鉴定出一种43（±2）kDa的蛋白质。从两个锌指到与真核转录因子氨基酸序列相似区域的Cre1蛋白片段，在大肠杆菌中作为与谷胱甘肽S-转移酶的融合蛋白表达。电泳迁移率变动分析（EMSA）和体外足迹分析表明融合蛋白与序列5'-GCGGAG-3'结合，该序列与构巢曲霉CreA结合的共有序列（5'-SYGGRG-3'）匹配良好。里氏木霉的无细胞提取物与携带该结合位点的DNA片段形成不同的复合物，并证明了这些复合物中存在Cre1和其他蛋白质。我们得出结论，里氏木霉Cre1是构巢曲霉CreA的功能同源物，并且它可能作为一种蛋白质复合物与其靶序列结合。
