Tschochne H, Milkereit P
BZH, Biochemie-Zentrum Heidelberg, Germany.
FEBS Lett. 1997 Jun 30;410(2-3):461-6. doi: 10.1016/s0014-5793(97)00636-4.
Terminated transcripts were generated at the ends of linearized DNA templates and at DNA-bound lac repressor by in vitro transcription with highly enriched or purified yeast RNA polymerase I (pol I). The release of the synthesized transcripts from the DNA was analyzed using immobilized DNA as template for the transcription reaction. An additional activity distinguishable from pol I was necessary to remove the terminated RNA from the template. Efficiency of transcript release could be improved if a thymidine-rich DNA fragment was located upstream of the transcriptional arrest caused by the DNA-bound lac repressor. The release activity interacted with different forms of polymerases, pol I able to initiate on the ribosomal gene promoter and pol I only active in non-specific transcription.
通过使用高度富集或纯化的酵母RNA聚合酶I(pol I)进行体外转录,在线性化DNA模板的末端以及与DNA结合的乳糖阻遏物处产生了终止转录本。使用固定化DNA作为转录反应的模板,分析了合成转录本从DNA上的释放情况。需要一种不同于pol I的额外活性来从模板上移除终止的RNA。如果富含胸腺嘧啶的DNA片段位于由与DNA结合的乳糖阻遏物引起的转录停滞上游,则转录本释放效率可以提高。释放活性与不同形式的聚合酶相互作用,一种是能够在核糖体基因启动子上起始的pol I,另一种是仅在非特异性转录中具有活性的pol I。