NRF-1, AN ACTIVATOR INVOLVED IN NUCLEAR-MITOCHONDRIAL INTERACTIONS, UTILIZES A NEW DNA-BINDING DOMAIN CONSERVED IN A FAMILY OF DEVELOPMENTAL REGULATORS
NRF-1, AN ACTIVATOR INVOLVED IN NUCLEAR-MITOCHONDRIAL INTERACTIONS, UTILIZES A NEW DNA-BINDING DOMAIN CONSERVED IN A FAMILY OF DEVELOPMENTAL REGULATORS
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DOI：
10.1101/gad.7.12a.2431
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发表时间：
1993-12-01
影响因子：
10.5
通讯作者：
SCARPULLA, RC
中科院分区：
文献类型：
--
作者：
VIRBASIUS, CMA;VIRBASIUS, JV;SCARPULLA, RC
核呼吸因子-1(NRF-1)最先被发现是细胞色素c基因的激活剂，随后被发现在核-线粒体相互作用中发挥更广泛的作用。我们现在已经克隆了一个编码NRF-1的HeLa基因，我们使用从胰酶多肽序列中提取的简并低聚物来进行PCR扩增。编码的蛋白在变性凝胶上与真正的HeLa细胞因子难以区分，表现出预期的NRF-1 DNA结合特异性，并在结合已知的NRF-1识别位点上与HeLa NRF-1具有相同的鸟嘌呤核苷酸接触。针对高度纯化的重组蛋白产生的抗血清识别使用粗核部分或近乎均一的HeLa NRF-1形成的相同的DNA-蛋白质复合体。重组的NRF-1还通过几个NRF-1反应启动子的特定位点激活转录，证实了该cDNA产物的转录活性和特异性。NRF-1的部分序列与海胆P3A2和果蝇的直立翅(EWG)蛋白密切相关。两者都是最近发现的发育调节因子。与P3A2和EWG序列同源性最高的区域在分子的氨基末端，通过缺失作图发现含有DNA结合区，而NRF-1的羧基末端与这两种蛋白质的序列差异很大。这些分子中的DNA结合域与DNA结合蛋白中常见的基序无关；因此，NRF-1、P3A2和EWG代表了一类新的高度保守的序列特异性调控因子的创始成员。
Nuclear respiratory factor 1 (NRF-1) was first discovered as an activator of the cytochrome c gene and was subsequently found to play a broader role in nuclear-mitochondrial interactions. We have now cloned a HeLa cDNA encoding NRF-1 using degenerate oligomers derived from tryptic peptide sequences for PCR amplification. The cDNA-encoded protein was indistinguishable from the authentic HeLa cell factor on denaturing gels, displayed the expected NRF-1 DNA-binding specificity, and made the same guanine nucleotide contacts as HeLa NRF-1 on binding known NRF-1 recognition sites. Antiserum raised against the highly purified recombinant protein recognized the identical DNA-protein complex formed using either a crude nuclear fraction or nearly homogeneous HeLa NRF-1. Recombinant NRF-1 also activated transcription through specific sites from several NRF-1-responsive promoters, confirming both the transcriptional activity and specificity of the cDNA product. Portions of NRF-1 are closely related to sea urchin P3A2 and the erect wing (EWG) protein of Drosophila. Both are recently identified developmental regulatory factors. The region of highest sequence identity with P3A2 and EWG was in the amino-terminal half of the molecule, which was found by deletion mapping to contain the DNA-binding domain, whereas the carboxy-terminal half of NRF-1 was highly divergent from both proteins. The DNA-binding domain in these molecules is unrelated to motifs found commonly in DNA-binding proteins; thus, NRF-1, P3A2, and EWG represent the founding members of a new class of highly conserved sequence-specific regulatory factors.