Abstract

The effects of GTP and ATP on the transcription termination and transcript production of bacteriophage T7 RNA polymerase were analyzed at the thr attenuator in the supercoiled and linearized DNA templates. T7 RNA polymerase and the supercoiled or linearized DNA template with the thr attenuator were preincubated with GTP or ATP first, then transcription was started by the addition of all four nucleotides. The results indicated that the termination efficiency of T7 RNA polymerase at the thr attenuator is significantly higher in the supercoiled DNA template than in the linearized one. With the preincubation of GTP, T7 RNA polymerase generates 4-21 times more RNA transcript in the supercoiled than linearized DNA template. Also, in the supercoiled DNA, T7 transcription with the preincubation of GTP produces 3.6-5.0 times more RNA transcript than that without preincubation. This strongly suggests that the topology of DNA template influences the transcript production of T7 RNA polymerase. Moreover, GTP may change the conformation of T7 RNA polymerase in the complex of transcription initiation.

中文摘要

在不同核酸形式下分析GTP和ATP對於噬菌體T7核醣核酸聚合轉錄終止和核酸產量的影響。T7核醣核酸聚合和帶有酥氨酸衰減子的不同形式核酸模板先和GTP或ATP作用，再加入四種核甘酸以啟動轉錄反應。結果發現在超螺旋狀核酸模板上，T7核醣核酸聚合對於酥氨酸衰減子所產生的轉錄終止效率，較在線形核酸模板上為高。並且T7核醣核酸聚合先和GTP作用者，在超螺旋狀核酸模板上所產生的核酸產量，較在線形核酸模板上高出4-21倍。同樣在超螺旋狀核酸模板上，T7核醣核酸聚合先和GTP作用者，所產生的核酸產量，較沒有先和GTP作用者多出3.6-5.0倍。這個結果顯示不同核酸形式會影響T7核醣核酸聚合的核酸產量，並且GTP會改變T7核醣核酸聚合在轉錄啟始時的構造。

Keyword: T7 RNA polymerase, Transcription termination, Supercoiled DNA.