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。

关键词:作用力;大肠杆菌启动子;RNA聚合酶;AFM;CAT酶活

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.

Key words:rupture force;E. coli promoter;RNA polymerase;AFM;CAT enzyme activity