Chen Y C, Jeng S T
Department of Botany, National Taiwan University, Taipei, Republic of China.
Biosci Biotechnol Biochem. 2000 Jun;64(6):1126-32. doi: 10.1271/bbb.64.1126.
A promoter competition assay was used to measure the stability of T7 RNA polymerase with its promoter. When T7 RNA polymerase was incubated with GTP for 5 minutes before the elongation of transcription in either the supercoiled or linearized template, the half-lives of T7 RNA polymerase-DNA complexes were reduced. The transcription product increased when T7 RNA polymerase preincubated with GTP in the supercoiled DNA but not in the linearized DNA template. On the other hand, preincubation of ATP with T7 RNA polymerase decreased the stability of T7 RNA polymerase with the supercoiled DNA, but did not affect the stability of T7 RNA polymerase with the linearized DNA. Furthermore, the production of RNA transcript was increased when T7 RNA polymerase was incubated with ATP in either supercoiled or linearized template before transcription elongation. This study is important to understand the relationship between the transcription initiation and the stability of the ternary complex, and to produce large quantities of RNA transcript in vitro.
采用启动子竞争分析来测定T7 RNA聚合酶与其启动子的稳定性。当在超螺旋或线性化模板上进行转录延伸之前，将T7 RNA聚合酶与GTP孵育5分钟时，T7 RNA聚合酶-DNA复合物的半衰期会缩短。当T7 RNA聚合酶在超螺旋DNA中与GTP预孵育时，转录产物增加，但在线性化DNA模板中则不然。另一方面，ATP与T7 RNA聚合酶预孵育会降低T7 RNA聚合酶与超螺旋DNA的稳定性，但不影响T7 RNA聚合酶与线性化DNA的稳定性。此外，在转录延伸之前，当T7 RNA聚合酶在超螺旋或线性化模板中与ATP孵育时，RNA转录本的产量会增加。这项研究对于理解转录起始与三元复合物稳定性之间的关系以及在体外大量生产RNA转录本具有重要意义。
