Burke C, Yu X B, Marchitelli L, Davis E A, Ackerman S
Biology Department, University of Massachusetts, Boston, MA 02125.
Nucleic Acids Res. 1990 Jun 25;18(12):3611-20. doi: 10.1093/nar/18.12.3611.
Eucaryotic transcription initiation by RNA polymerase II involves protein:DNA interactions during the formation of a transcription complex. In addition to RNA polymerase II there are at least five other general transcription factors necessary for initiation with the adenovirus major late promoter. One of these, TFIIA, is involved in the earliest events during transcription complex assembly. We have purified TFIIA from wheat germ and characterized it in an in vitro transcription system. Wheat TFIIA is a single polypeptide of Mr approximately 35 kd which functionally replaces human (HeLa) TFIIA to form a wheat/HeLa transcription system. [This polypeptide can be eluted from a SDS-polyacrylamide gel, refolded to a native conformation, and will function as wheat TFIIA in the heterologous system.] The heterologous system requires a lower optimal incubation temperature than the HeLa system. Biochemical characterization, using the adenovirus major late promoter, indicates that transcription reaction parameters for both wheat and HeLa TFIIA are similar but the kinetics of transcription for both TFIIAs are somewhat dissimilar. A plant viral promoter, the cauliflower mosaic virus 35S promoter, accurately and efficiently directs in vitro transcription in both the wheat/HeLa and HeLa systems with identical transcription kinetics. We conclude that TFIIA function has been conserved during evolution.
RNA聚合酶II介导的真核转录起始在转录复合物形成过程中涉及蛋白质与DNA的相互作用。除RNA聚合酶II外，腺病毒主要晚期启动子起始转录至少还需要其他五种通用转录因子。其中之一TFIIA参与转录复合物组装过程中最早的事件。我们从小麦胚芽中纯化了TFIIA，并在体外转录系统中对其进行了表征。小麦TFIIA是一条分子量约为35kd的单链多肽，在功能上可替代人（HeLa）TFIIA，从而形成一个小麦/HeLa转录系统。[这条多肽链可从SDS聚丙烯酰胺凝胶上洗脱下来，重新折叠成天然构象，并在异源系统中发挥小麦TFIIA的功能。]该异源系统所需的最佳孵育温度低于HeLa系统。利用腺病毒主要晚期启动子进行的生化表征表明，小麦和HeLa TFIIA的转录反应参数相似，但两种TFIIA的转录动力学有所不同。一种植物病毒启动子，即花椰菜花叶病毒35S启动子，在小麦/HeLa和HeLa系统中均能准确、高效地指导体外转录，且转录动力学相同。我们得出结论，TFIIA的功能在进化过程中得以保留。
