Paluh J L, Yanofsky C
Department of Biological Sciences, Stanford University, California 94305.
Mol Cell Biol. 1991 Feb;11(2):935-44. doi: 10.1128/mcb.11.2.935-944.1991.
CPC1 is the transcriptional activator of amino acid biosynthetic genes of Neurospora crassa. CPC1 function in vivo was abolished upon deletion of segments of cpc-1 corresponding to the presumed transcription activation domain, the DNA-binding and dimerization domains, or a 52-residue connector segment of CPC1. A truncated CPC1 polypeptide containing only the carboxy-terminal 57-residue segment of CPC1 was sufficient to form homodimers that bound DNA. However, deletion of the segment of cpc-1 corresponding to the connector segment in the full-length CPC1 polypeptide abolished DNA binding. Removal of a segment of cpc-1 corresponding to the GIn-rich region of CPC1 reduced in vivo function only slightly. The homologous transcription activator of Saccharomyces cerevisiae, GCN4, did not substitute for CPC1 in N. crassa. Chimeric CPC1-GCN4 polypeptides that contained the GCN4 transcriptional activation domain or the domain of GCN4 that corresponds to the essential 52-residue connector segment of CPC1, functioned with reduced efficiency. However, a chimeric polypeptide containing the GCN4 DNA-binding and dimerization domains in place of those of CPC1 functioned essentially as well as wild-type CPC1. The basic and dimerization domains of CPC1 were characterized by introducing deletions or site-directed amino acid replacements. The basic region was required for DNA binding but not for dimerization. CPC1 has a short dimerization domain containing heptad residues Leu-1, Leu-2, Trp-3, and His-4. When Val was substituted for Leu-1 or Leu-2, CPC1 was fully active, but when Val replaced Trp-3, dimerization and DNA binding were prevented. DNA band shift analyses with CPC1 heterodimers demonstrated that CPC1 does not require aligned heptad leucine residues for dimerization. Replacement of two charged residues located between Leu-1 and Leu-2 of CPC1 abolished dimerization and DNA binding.
CPC1是粗糙脉孢菌氨基酸生物合成基因的转录激活因子。当删除cpc - 1中对应于推测的转录激活结构域、DNA结合和二聚化结构域或CPC1的52个残基连接片段的部分时，CPC1在体内的功能丧失。仅包含CPC1羧基末端57个残基片段的截短CPC1多肽足以形成结合DNA的同型二聚体。然而，删除全长CPC1多肽中对应于连接片段的cpc - 1片段会消除DNA结合。删除cpc - 1中对应于CPC1富含谷氨酰胺区域的片段只会略微降低其体内功能。酿酒酵母的同源转录激活因子GCN4不能替代粗糙脉孢菌中的CPC1。包含GCN4转录激活结构域或对应于CPC1必需的52个残基连接片段的GCN4结构域的嵌合CPC1 - GCN4多肽功能效率降低。然而，用GCN4的DNA结合和二聚化结构域取代CPC1的相应结构域的嵌合多肽的功能基本上与野生型CPC1相同。通过引入缺失或定点氨基酸替换对CPC1的碱性和二聚化结构域进行了表征。碱性区域是DNA结合所必需的，但不是二聚化所必需的。CPC1有一个短的二聚化结构域，包含七肽残基Leu - 1、Leu - 2、Trp - 3和His - 4。当用Val取代Leu - 1或Leu - 2时，CPC1完全有活性，但当Val取代Trp - 3时，二聚化和DNA结合被阻止。用CPC1异源二聚体进行的DNA条带迁移分析表明，CPC1二聚化不需要七肽亮氨酸残基对齐。替换位于CPC1的Leu - 1和Leu - 2之间的两个带电荷残基会消除二聚化和DNA结合。
