Analysis Of Interaction Between Escherichia Coli Promoter And Rna Polymerase By Afm Force Spectroscopy

Promoter,as an important cis-regulatory element in gene expression,regulates protein expression by controlling gene transcription.In microbial metabolic engineering,it has become a commonly used strategy for metabolic transformation in industrial microbiology to achieve the optimal accumulation of desired products by changing promoter strength and regulating metabolic flow distribution.In the field of metabolic engineering and synthetic biology,the standardization and modularization of metabolic regulatory elements have become the development trend,therefore,efficient and accurate quantitative promoter characterization is needed to establish strategies for rapid screening of promoters.Due to the interference of other regulatory elements,the influence of cellular physiological state and cumbersome operation in the currently common used characterization method based on reporter gene expression,it is urgent to establish a direct and efficient method for quantitative analysis of promoters.With phage T7-based transcription system as a model system,this study firstly investigated the interaction between T7 promoter and RNA polymerase(RNAp)by Atomic Force Microscope(AFM)force spectroscopy,and established an in vitro analysis platform to investigate the promoter and RNAp interaction.The efficacy of in vitro interaction analysis for promoter characterization was verified via in vivo analysis and series of control experiments.Based on this platform,with promoters of Escherichia coli as subjects,a series of promoters with mutations at key position were invesitigated.This study for the first draw the correlation between promoter sequence and activity via the promoter/RNAp interaction analysis in vitro.The main results of this study are as follows:(1)Establishing a strategy for RNAp and promoter immobilization for AFM force spectroscopy analysis.The optimal modification strategy for the alkylation of substrate was selected by AFM images of substrates modified by different processes;and the T7 RNAp concentration used for silicon substrates immobilization was optimized.Finally,a silicon substrate immobilized with RNAp,which is uniform distributed and with moderate density.Meanwhile,promoters were immobilized on the surface of the AFM probe by Au-s coordination bond formed between mercapto groups at the 3'end of the promoter sequence and the gold plating surface of the probe.Verified by multiple control groups,the established method for promoter/T7 RNAp interaction system reduced the probability of nonspecific binding and improved the sensitivity of the AFM force spectroscopy.(2)Based on AFM force spectroscopy,the method for investigation the interaction between promoters and RNAp in vitro was established.The interaction force(304.2±12.7 pN)between T7 promoter and T7 RNAp was obtained by AFM force-distance curve combined with statistical analysis.Cofirmed by control experiments,the interaction obtained was caused by the specific binding between T7 promoter and RNAp.Mutation at-10 and-11 sites in T7 promoter was applied to measure the interaction force and binding probability between mutant promoter and T7 RNAp.The above promoters were characterized by the enzyme activity of Chloramphenicol Acetyl transferase(CAT)using the promoter-probe plasmid,which was the product of the reporter gene cat,and the results were consistent with that chatacterized in vitro by AFM force spectroscopy.These results verified the validity of in vitro promoter interaction analysis By AFM force spectroscopy.(3)Screening of promoters in Escherichia coli based on AFM force spectroscopy.The optimal concentration for the immobilization of Escherichia coli RNAp substrate was determined to be 100 U·m L-1.Based on the AFM force spectroscopy analysis of promoter strength established in the former research,the Ls1 promoter in Escherichia coli with high activity was selected as the original sequence,and a series of mutants at key sites were synthesized.The interaction force between promoters and RNAp,and the binding probabilities were investigated.Furthermore,the activity of promoters were verified by determining the activity of CAT enzyme via the promoter probe plasmid.(4)Correlation analysis between promoter sequence of Escherichia coli and interaction information of promoter/RNAp obtained in vitro.Ls1 was reported as a strong promoter with conserved sequences in the-10 and-35 regions.The rupture force between Ls1 and RNAp was analyzed by AFM force specotroscopy and determined as 331.1±5.1 pN.Ls2 promoter,which was lack of-10 region of Ls1 promoter,was desinged.The in vitro analysis of its interaction revealed that the histogram distribution of the rupture force did not presente characteristics of normal distribution and siginificantly less binding events compared with Ls1 promoter.With conserved sequences in the-10 region as Ls1 promoter,Ls1-T2(1 base differences),Ls1-T1(1 base differences)and Ls1-35 promoters(3 base differences)mutants with mutation at-35 region were obtained.The interaction forces were measured as 293.3±5.5 pN,271.9±6.7 pN and 243.5±5.2 pN for Ls1-T2,Ls1-T1 and Ls1-35 promoters respectively.Compared with Ls1 promoter,it was found that the more conservative the sequence at key postions,the larger rupture force it requires to break down the promoter/RNAp complex.These results for the first time correlated the sequence characteristic of the promoter with the perspective of molecular interaction between promoter and RNAp,and demonstrated that the efficacy of promoter characterization via in vitro analysis by AFM force spectroscopy.