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Analysis of premature termination in c-myc during transcription by RNA polymerase II in a HeLa nuclear extract.

机译：在HeLa核提取物中通过RNA聚合酶II转录过程中c-myc的过早终止的分析。

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Transcriptional regulation of the human c-myc gene, an important aspect of cellular differentiation, occurs in part at the level of transcript elongation. In vivo, transcriptional arrest, due to either pausing or termination, occurs near the junction between the first exon and first intron and varies with the growth state of the cell. We have tested the transcription of c-myc templates in HeLa nuclear extracts. We did not observe significant arrest under standard conditions, but we found that a considerable fraction of transcription complexes stopped at the c-myc TII site (just past the first exon-intron junction) when the KCl concentration was raised to 400 mM during elongation. Transcriptional arrest at TII also was observed at KCl concentrations as low as 130 mM and when potassium acetate or potassium glutamate was substituted for KCl. Under these conditions, arrest occurred at the TII site when transcription was initiated at either the c-myc P2 promoter or the adenovirus 2 major late promoter. Further, the TII sequence itself, in forward but not reverse orientation, was sufficient to stop transcription in a HeLa nuclear extract. By separating the TII RNA from active transcription complexes by using gel filtration, we found that arrest at TII at 400 mM KCl resulted in transcript release and thus true transcriptional termination. The efficiency of termination at TII depended on the growth state of the cells from which the extracts were made, suggesting that some factor or factors control premature termination in c-myc.
机译：人类c-myc基因的转录调节是细胞分化的重要方面，部分在转录本延伸水平上发生。在体内，由于暂停或终止，转录停滞发生在第一外显子和第一内含子之间的连接附近，并随着细胞的生长状态而变化。我们已经测试了HeLa核提取物中c-myc模板的转录。我们在标准条件下没有观察到明显的停滞现象，但是我们发现当延伸过程中KCl浓度升高到400 mM时，相当一部分转录复合物停止在c-myc TII位点（刚好超过第一个外显子-内含子连接处）。在KCl浓度低至130 mM以及用乙酸钾或谷氨酸钾替代KCl时，也观察到TII处的转录停滞。在这些条件下，当在c-myc P2启动子或腺病毒2主要晚期启动子处开始转录时，在TII位点发生逮捕。此外，TII序列本身（正向而非反向）足以终止HeLa核提取物中的转录。通过使用凝胶过滤从活性转录复合物中分离TII RNA，我们发现在400 mM KCl的TII处停滞会导致转录物释放，从而导致真正的转录终止。在TII处终止的效率取决于提取物所制成的细胞的生长状态，表明某些因素控制c-myc中的过早终止。

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