细菌转录起始调控机制*

原核生物同一种群的每个细胞都是和外界环境直接接触的,它们主要通过开启或关闭某些基因的表达来适应环境条件。所以,环境因子往往是调控的效应因子,必须严格调控转录来确保细胞对环境改变做出有效且充分的反应。原核生物基因的表达受多种因素的调控,而对于大多数细菌来说,调控基因表达的关键步骤是启动子识别和RNA聚合酶启动转录。在细菌的细胞中,可以通过调节RNA聚合酶的活性以及改变RNA聚合酶对启动子的结合来优化基因的转录过程以适应不同环境变化。总结了目前已发现的参与细菌细胞转录调节的各类因子,从这些因子对启动子的作用、RNA聚合酶的作用以及两者的相互作用等方面阐述它们调控基因表达的分子机制。总结多种基因调控的作用,加深对转录起始过程的认识,希望能对未来调控转录起始过程来实现目标基因的高效表达和不利基因的抑制表达提供思路,为以后的工业菌株改造提供依据。     

Rapid binding of T7 RNA polymerase is followed by simultaneous bending and opening of the promoter DNA

To form a functional open complex, bacteriophage T7 RNA polymerase (RNAP) binds to its promoter DNA and induces DNA bending and opening. The objective of this study was to elucidate the temporal coupling in DNA binding, bending, and opening processes that occur during initiation. For this purpose, we conducted a combined measurement of stopped-flow fluorescence anisotropy, fluorescence resonance energy transfer (FRET), and 2-aminopurine fluorescence. Stopped-flow anisotropy measurements provided direct evidence of an intermediate resulting from rapid binding of the promoter to T7 RNA polymerase. Stopped-flow FRET measurements showed that promoter bending occurred at a rate constant that was slower than the initial DNA binding rate constant, indicating that the initial complex was not significantly bent. Similarly, stopped-flow 2-aminopurine fluorescence changes showed that promoter opening occurred at a rate constant that was slower than the initial DNA binding rate constant, indicating that the initial complex was not significantly melted. The indistinguishable observed rate constants of FRET and 2-aminopurine fluorescence changes indicate that DNA bending and opening processes are temporally coupled and these DNA conformational changes take place after the DNA binding step. The results in this paper are consistent with the mechanism in which the initial binding of T7 RNAP to the promoter results in a closed complex, which is then converted into an open complex in which the promoter is both sharply bent and melted.