Purification and initial characterization of RNA polymerase from Thermus thermophilus strain HB8.
Purification and initial characterization of RNA polymerase from Thermus thermophilus strain HB8.
复制标题
嗜热栖热菌 HB8 菌株 RNA 聚合酶的纯化和初步表征。
作者：
Xue,Y;Hogan,BP;Erie,DA
采用一种新的、快速的双柱纯化方法，在不到2天的时间内，从嗜热菌Thermus thermophilusHB8中获得了DNA依赖的RNA聚合酶(RNAP)，其纯度可达65%(RNAP活性测定)。用含有λPR启动子的DNA对纯化的酶进行了鉴定。KMnO4足迹、失败的起始试验和特定停滞伸长复合体的形成提供了令人信服的证据。嗜热菌RNA聚合酶可以与含有λPR启动子的DNA结合，形成开放的复合体，并以依赖于温度的方式启动转录。这一证据表明。嗜热菌RNAP具有较小的内禀结合能。ColiRNAP。相反，嗜热梭菌依赖于其环境的高温来提供所需的热能，以刺激开放启动子复合体的形成，启动转录，并促进RNA聚合酶的构象变化，从而导致核苷酸掺入。
Utilizing a novel and rapid two-column purification procedure, the DNA-dependent RNA polymerase (RNAP) from the thermophile,Thermus thermophilusHB8, was purified to electrophoretic homogeneity with a recovery of 65% (as determined by RNAP activity) in less than 2 days. The purified enzyme was characterized using DNA containing the λPRpromoter. KMnO4footprinting, abortive initiation assays, and the formation of the specific stalled elongation complex provide compelling evidence thatT. thermophilusRNA polymerase can bind to DNA containing the λPRpromoter, form an open complex, and initiate transcription in a temperature-dependent manner. This evidence suggests thatT. thermophilusRNAP possesses less intrinsic binding energy thanE. coliRNAP. Instead,T. thermophilusrelies on the high temperatures of its environment to provide the thermal energy required to stimulate open promoter complex formation, initiate transcription, and facilitate the conformational changes in RNA polymerase that result in nucleotide incorporation.