Fuhrman S A, Engelke D R, Geiduschek E P
J Biol Chem. 1984 Feb 10;259(3):1934-43.
That stable transcription complexes are formed in HeLa cell RNA polymerase III transcription extracts (Weil, P. A., Segall, J., Harris, B., Ng, S.-Y., and Roeder, R. G. (1979) J. Biol. Chem. 254, 6163-6173) can be shown in order of template addition experiments with DNAs coding for adenovirus 2 VA I RNA or one of the Bombyx mori tRNAAla2 genes. Using this property of the HeLa extracts in a "second template rescue assay" has allowed us to partially purify protein components involved in stable transcription complex formation. Two fractions, called transcription fraction X (TfrX) and transcription fraction Y (TfrY), are required to fully reconstitute selective transcription of the VA I and tRNAAla2 DNA templates. TfrX contains one or more components required for forming transcriptional pre-emptive complexes, as shown in order of addition experiments. TfrX strongly protects a DNA segment surrounding the highly conserved distal sequence (the so-called B block) of the VA I and tRNAAla2 genes from DNase I digestion; we have also characterized weak protection of other segments of these genes by TfrX. DNase I protection experiments with TfrX and probes prepared from deletion variants of the VA I gene show that an intact B block is required for the strong protection.
在HeLa细胞RNA聚合酶III转录提取物中可形成稳定的转录复合物（Weil, P. A., Segall, J., Harris, B., Ng, S.-Y., and Roeder, R. G. (1979) J. Biol. Chem. 254, 6163 - 6173），这可以通过对编码腺病毒2 VA I RNA或家蚕tRNAAla2基因之一的DNA进行模板添加实验来证明。利用HeLa提取物的这一特性，在“第二次模板拯救试验”中，我们得以部分纯化参与稳定转录复合物形成的蛋白质成分。需要两个组分，即转录组分X（TfrX）和转录组分Y（TfrY），才能完全重建VA I和tRNAAla2 DNA模板的选择性转录。如添加顺序实验所示，TfrX包含形成转录抢先复合物所需的一种或多种成分。TfrX能强烈保护VA I和tRNAAla2基因高度保守的远端序列（即所谓的B区）周围的DNA片段不被DNase I消化；我们还对TfrX对这些基因其他片段的弱保护作用进行了表征。用TfrX和从VA I基因缺失变体制备的探针进行的DNase I保护实验表明，完整的B区是强保护所必需的。
