Cabrejos M E, Maldonado E
Programa de Biologia Celular Molecular, Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago.
IUBMB Life. 2000 Aug;50(2):125-9. doi: 10.1080/713803697.
The aim of this work was to identify proteins from Adenovirus 2-infected HeLa cell extracts that interact with the carboxyl-terminal domain of the largest subunit of RNA polymerase II. First, a mammalian RNA polymerase II complex was isolated from Adenovirus 2-infected HeLa cell extracts by affinity chromatography against the carboxyl-terminal domain of the largest subunit of RNA polymerase II, followed by chromatography on a Mono S fast protein liquid chromatographic column. Second, the isolated complex was further characterized by Western blot analysis, the formation of a GMP-protein complex, and transcriptional activity. The isolated complex contains general transcription factors, chromatin-remodeling factors, histone acetyltransferases, Srbs, capping enzymes, and E1A viral oncoproteins. The RNA polymerase II complex is active in transcription when supplemented with recombinant transcription factor IIB.
这项工作的目的是从腺病毒2感染的HeLa细胞提取物中鉴定与RNA聚合酶II最大亚基的羧基末端结构域相互作用的蛋白质。首先,通过针对RNA聚合酶II最大亚基的羧基末端结构域的亲和色谱法,从腺病毒2感染的HeLa细胞提取物中分离出哺乳动物RNA聚合酶II复合物,然后在Mono S快速蛋白质液相色谱柱上进行色谱分离。其次,通过蛋白质印迹分析、GMP-蛋白质复合物的形成和转录活性对分离出的复合物进行进一步表征。分离出的复合物包含一般转录因子、染色质重塑因子、组蛋白乙酰转移酶、Srbs、加帽酶和E1A病毒癌蛋白。当补充重组转录因子IIB时,RNA聚合酶II复合物在转录中具有活性。