DNA形式對於RNA產量及轉錄終止的影響

Abstract

不論?核或原核生物，DNA都是以negative supercoil的形式存在細胞中，supercoil的形式已被報告會影響E. coli RNA polymerase的轉錄作用及核中蛋白質與DNA的結合，也就是說，DNA的形式會影響基因的表現。 在這篇研究中，我們使用了19 thr--包含了T7?動子及rho-independent-like終止子-- 這個質體作為範本，來分析探討DNA形式（topology）對於T7 RNA polymerase離體轉錄時，RNA產量及終止的效率的影響。將Supercoil與被限制酵素切成線性的DNA做比較則可以發現：T7 RNA polymeraes在Supercoil DNA作為範本時，在終止子的位置會有較高的終止效率，同樣的，當四種NTP其中任一種濃度限制在5μM時，以supercoil DNA當作範本的反應會有較高的產量。然而，當NTP濃度高於5μM時，以線性化DNA作為範本的反應則有比較高的RNA產量。所以，我們證明瞭DNA的形式明顯地影響了T7 RNA polymerase的終止效率與RNA產量。 更進一步地，我們將DNA範本與GTP、T7 RNA polymerase先置於37℃ 5分鐘，再加入其他的NTP開始進行轉錄作用，我們發現：以supercoiled DNA作為範本的反應，RNA產量是線性化範本的3-5倍。這樣的現象在先加入ATP時並沒有發生。這可能指出：特別當supercoil DNA在只含GTP時，T7 RNA polymerase會形成有很高活性的轉錄起始複合體，當同時有其他NTP存在時，會與GTP彼此競爭而降低形成高活性轉錄起始複合體的機會。

In both eukaryote and prokaryote, most DNA within cells is present as negative supercoil. The presence of DNA supercoiling has been reported to affect the transcripiton of E. coli RNA polymerase and DNA binding of nuclear proteins, and suggested to be related to gene regulation. In this study, in order to analyze the effects of DNA topology on transcription and termination of T7 RNA polymerase in vitro, a DNA template containing a T7 promoter and rho-independent-like terminator is used. When supercoiled DNA and DNA linearized by restriction enzyme were compared, supercoiled DNA can enhance the transcription termination of T7 RNA polymerase at this rho-independent terminator. Also, T7 RNA polymerase produces more transcript on supercoil DNA template than linearized template when any one of the four nucleotide triphosphates (NTPs) supplied to the reaction is 5 μM. As the concentration of NTPs in reaction is greater than 5 μM, more transcript is produced in the linearized DNA than supercoiled DNA by T7 RNA polymerase. Therefore, DNA topology do influence the transcription and termination of T7 RNA polymerase. Furthermore, DNA template was preincubated with T7 RNA polymerase and GTP, but not ATP, at 37℃ for 5 minutes, before NTPs were added to start the transcription. T7 RNA polymerase with supercoil DNA template produces three to five times more transcript than with linearized template. This may imply that especially in the supercoiled DNA template GTP alone can help T7 RNA polymerase to form active transcription initiation complex. In the presence of all four NTPs, GTP may compete with other NTPs in the open promoter complex, and consequently T7 RNA polymerase decreases the possibility of forming active transcription initiation complex.