The specific recognition and binding of promoter and RNA polymerase is the first step of transcription initiation in bacteria and largely determines transcription activity. Therefore, direct analysis of the interaction between promoter and RNA polymerase in vitro may be a new strategy for promoter characterization, to avoid interference due to the cell's biophysical condition and other regulatory elements. In the present study, the specific interaction between T7 promoter and T7 RNA polymerase was studied as a model system using force spectroscopy based on atomic force microscope (AFM). The specific interaction between T7 promoter and T7 RNA polymerase was verified by control experiments, and the rupture force in this system was measured as 307.2 ± 6.7 pN. The binding between T7 promoter mutants with various promoter activities and T7 RNA polymerase was analyzed. Interaction information including rupture force, rupture distance and binding percentage were obtained in vitro, and reporter gene expression regulated by these promoters was also measured according to a traditional promoter activity characterization method in vivo. Using correlation analysis, it was found that the promoter strength characterized by reporter gene expression was closely correlated with rupture force and the binding percentage by force spectroscopy. These results indicated that the analysis of the interaction between promoter and RNA polymerase using AFM-based force spectroscopy was an effective and valid approach for the quantitative characterization of promoters.
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January 2018
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A 3D representation of the filamentous cyanobacteria Anabaena. In this issue, Sein-Echaluce et al. report on the molecular basis for the integration of environmental signals by FurB from Anabaena sp. PCC 7120; for details see pages 151–168.
Research Article|
January 11 2018
Investigation of specific interactions between T7 promoter and T7 RNA polymerase by force spectroscopy using atomic force microscope
Xiaojuan Zhang;
Xiaojuan Zhang
1National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China
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Zhixuan Yao;
Zhixuan Yao
2School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China
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Yanting Duan;
Yanting Duan
2School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China
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Xiaomei Zhang;
Xiaomei Zhang
3School of Pharmaceutical Science, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China
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Jinsong Shi;
Jinsong Shi
3School of Pharmaceutical Science, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China
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Zhenghong Xu
1National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China
2School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China
Correspondence: Zhenghong Xu (zhenghxu@jiangnan.edu.cn)
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Biochem J (2018) 475 (1): 319–328.
Article history
Received:
August 07 2017
Revision Received:
November 23 2017
Accepted:
November 29 2017
Accepted Manuscript online:
November 29 2017
Citation
Xiaojuan Zhang, Zhixuan Yao, Yanting Duan, Xiaomei Zhang, Jinsong Shi, Zhenghong Xu; Investigation of specific interactions between T7 promoter and T7 RNA polymerase by force spectroscopy using atomic force microscope. Biochem J 15 January 2018; 475 (1): 319–328. doi: https://doi.org/10.1042/BCJ20170616
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