Jackson D A, Iborra F J, Manders E M, Cook P R
Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, United Kingdom.
Mol Biol Cell. 1998 Jun;9(6):1523-36. doi: 10.1091/mbc.9.6.1523.
Using HeLa cells, we have developed methods to determine 1) the number of RNA polymerases that are active at any moment, 2) the number of transcription sites, and 3) the number of polymerases associated with one transcription unit. To count engaged polymerases, cells were encapsulated in agarose, permeabilized, treated with ribonuclease, and the now-truncated transcripts extended in [32P]uridine triphosphate; then, the number of growing transcripts was calculated from the total number of nucleotides incorporated and the average increment in length of the transcripts. Approximately 15, 000 transcripts were elongated by polymerase I, and approximately 75,000 were elongated by polymerases II and III. Transcription sites were detected after the cells were grown in bromouridine for <2.5 min, after which the resulting bromo-RNA was labeled with gold particles; electron microscopy showed that most extranucleolar transcripts were concentrated in approximately 2400 sites with diameters of approximately 80 nm. The number of polymerases associated with a transcription unit was counted after templates were spread over a large area; most extranucleolar units were associated with one elongating complex. These results suggest that many templates are attached in a "cloud" of loops around a site; each site, or transcription "factory," would contain approximately 30 active polymerases and associated transcripts.
利用海拉细胞，我们开发了一些方法来确定：1）任一时刻活跃的RNA聚合酶数量；2）转录位点的数量；3）与一个转录单元相关的聚合酶数量。为了计数参与转录的聚合酶，将细胞包埋在琼脂糖中，使其通透化，用核糖核酸酶处理，然后用[32P]尿苷三磷酸延伸现在已截短的转录本；接着，根据掺入的核苷酸总数和转录本长度的平均增量来计算正在延伸的转录本数量。约15000个转录本由聚合酶I延伸，约75000个转录本由聚合酶II和III延伸。在细胞于溴尿苷中培养不到2.5分钟后检测转录位点，之后用金颗粒标记产生的溴化RNA；电子显微镜显示，大多数核仁外转录本集中在直径约80纳米的约2400个位点。在模板大面积铺展后计数与一个转录单元相关的聚合酶数量；大多数核仁外单元与一个延伸复合物相关。这些结果表明，许多模板以环状“云”的形式附着在一个位点周围；每个位点，即转录“工厂”，将包含约30个活跃的聚合酶及相关转录本。
