Discipline of Biological Sciences, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW, Australia.
Protein Sci. 2009 Nov;18(11):2287-97. doi: 10.1002/pro.239.
RNA polymerase (RNAP) is an essential and highly conserved enzyme in all organisms. The process of transcription initiation is fundamentally different between prokaryotes and eukaryotes. In prokaryotes, initiation is regulated by sigma factors, making the essential interaction between sigma factors and RNAP an attractive target for antimicrobial agents. Our objective was to achieve the first step in the process of developing novel antimicrobial agents, namely to prove experimentally that the interaction between a bacterial RNAP and an essential sigma factor can be disrupted by introducing carefully designed mutations into sigma(A) of Bacillus subtilis. This disruption was demonstrated qualitatively by Far-Western blotting. Design of mutant sigmas was achieved by computer-aided visualization of the RNAP-sigma interface of the B. subtilis holoenzyme (RNAP + sigma) constructed using a homology modeling approach with published crystal structures of bacterial RNAPs. Models of the holoenzyme and the core RNAP were rigorously built, evaluated, and validated. To allow a high-quality RNAP-sigma interface model to be constructed for the design of mutations, a crucial error in the B. subtilis sigma(A) sequence in published databases at amino acid 165 had to be corrected first. The new model was validated through determination of RNAP-sigma interactions using targeted mutations.
RNA 聚合酶（RNAP）是所有生物中一种必不可少且高度保守的酶。原核生物和真核生物的转录起始过程在根本上有所不同。在原核生物中，起始受σ因子调节，因此，σ因子与 RNAP 之间的基本相互作用成为了抗菌药物的一个有吸引力的靶点。我们的目标是实现开发新型抗菌药物过程中的第一步，即通过在枯草芽孢杆菌σ(A)中引入精心设计的突变，从实验上证明细菌 RNAP 和必需的σ因子之间的相互作用可以被破坏。这种破坏通过 Far-Western 印迹进行了定性证明。突变σ因子的设计是通过计算机辅助可视化枯草芽孢杆菌全酶（RNAP+σ）中的 RNAP-σ 界面来实现的，该全酶是使用已发表的细菌 RNAP 晶体结构的同源建模方法构建的。全酶和核心 RNAP 的模型被严格构建、评估和验证。为了能够为突变设计构建高质量的 RNAP-σ 界面模型，首先必须纠正发表的数据库中枯草芽孢杆菌σ(A)序列在氨基酸 165 处的一个关键错误。通过使用靶向突变来确定 RNAP-σ 相互作用来验证新模型。
