Gabrielsen O S, Marzouki N, Ruet A, Sentenac A, Fromageot P
Département de Biologie, Centre d'Etudes Nucléaires de Saclay, Gif-sur-Yvette, France.
J Biol Chem. 1989 May 5;264(13):7505-11.
Yeast transcription factor tau interacts with the A and B blocks of the intragenic promoter of tRNA genes. The structure of tau was investigated by identifying the polypeptide chains specifically complexed to the tRNA3Glu gene. Highly purified factor, obtained by an improved purification procedure, contained several polypeptide chains, four of which (Mr = 145,000, 135,000, 100,000 and 65,000) comigrated with tau-DNA complex by polyacrylamide gel electrophoresis. Antibodies raised against the 145- and 100-kDa components altered the migration of tau-DNA complexes in band shift assays and inhibited tRNA synthesis in a reconstituted transcription system. These components are immunologically unrelated proteins. By UV cross-linking to 32P-body-labeled tDNA followed by extensive DNase treatment, two polypeptides of the same size (145 and 100 kDa) were found to be radioactively labeled. Factor tau, therefore, appears to be a multisubunit DNA-binding protein with two distinct polypeptides contributing to DNA recognition. Limited proteolysis of tau generated a protease-resistant tau B (tau B) domain that binds solely to the B block. tau B-tDNA complexes were recognized by anti-145 IgG and contained a 120-kDa polypeptide that could originate from the 145-kDa component by proteolysis. These results strongly suggest that the 145-kDa polypeptide belongs to tau B and is responsible for B block binding.
酵母转录因子tau与tRNA基因基因内启动子的A和B区段相互作用。通过鉴定与tRNA3Glu基因特异性结合的多肽链来研究tau的结构。通过改进的纯化程序获得的高度纯化的因子包含几条多肽链，其中四条（分子量分别为145,000、135,000、100,000和65,000）在聚丙烯酰胺凝胶电泳中与tau-DNA复合物迁移一致。针对145 kDa和100 kDa组分产生的抗体在凝胶迁移实验中改变了tau-DNA复合物的迁移，并在重组转录系统中抑制了tRNA合成。这些组分是免疫无关的蛋白质。通过对32P-体内标记的tDNA进行紫外线交联，然后进行广泛的DNase处理，发现两条大小相同（145 kDa和100 kDa）的多肽被放射性标记。因此，因子tau似乎是一种多亚基DNA结合蛋白，有两种不同的多肽参与DNA识别。对tau进行有限的蛋白酶解产生了一种仅与B区段结合的蛋白酶抗性tau B（tau B）结构域。tau B-tDNA复合物可被抗145 IgG识别，并含有一条120 kDa的多肽，该多肽可能是由145 kDa组分经蛋白酶解产生的。这些结果强烈表明，145 kDa多肽属于tau B，并负责与B区段结合。
