原子力显微镜力谱研究大肠杆菌启动子与RNA聚合酶的相互作用

Atomic force microscope based force spectroscopy as tool to study the interaction between Escherichia coli promoter and RNA polymerase

摘要:

[目的]本研究通过原子力显微镜（AFM）力谱技术研究了大肠杆菌启动子与RNA聚合酶（RNAp）间的相互作用，目的是建立一种高效的体外表征启动子的新方法。[方法]优化了用于单分子AFM力谱分析的蛋白固定化策略，建立AFM力谱分析启动子的策略，以缺失识别启动子序列的σ亚基核心RNA聚合酶（RNAp-C）为对照，研究启动子/RNAp间相互作用的特异性。最后比较了序列较典型的Ls1启动子和缺失–10区的Ls2启动子的力谱。[结果]基于建立的方法，验证了Ls1与大肠杆菌RNAp结合的特异性，其相互作用力为（331.10±5.10） pN。与Ls1相比，Ls2启动子与RNAp结合显著减少。利用启动子探针质粒，以报告基因cat的表达产物氯霉素乙酰转移酶（CAT）的酶活验证Ls1、Ls2启动子强度，分别为（181.70±4.10）、（0.30±0.20） U/mg。[结论]本研究建立的基于AFM力谱技术的启动子分析技术，是一种高效的、直接定量表征启动子活性的新方法。

Abstract:

[Objective] To establish an efficient method for promoter characterization, the interaction between E. coli promoter and RNA polymerase (RNAp) was studied by atomic force microscope (AFM) based force spectroscopy.[Methods] Protein immobilization was optimized, and the specific promoter/RNAp interaction was verified using core-RNAp (RNAp-C) as control. The force spectrum of promoter Ls2, lack of -10 region of Ls1, was studied.[Results] Based on the established method, the specific interaction between promoter Ls1 and RNAp was studied, and the rupture force was measured as (331.10±5.14) pN. Ls2 showed significantly less binding events towards RNAp compared with Ls1. Using promoter-probe plasmid, the activities of Ls1 and Ls2 were verified by reporter gene-cat, which were (181.73±4.08) U/mg and (0.33±0.21) U/mg, respectively.[Conclusion] A novel promoter analysis method based on AFM force spectroscopy was established. The results demonstrated this method can be applied in quantitative characterization of promoter with high efficacy and reliability.