Establishment Of Escherichia Coli Terminator Characterization Strategy And Analysis Of Regulatory Functions

The terminator is a special hairpin structure,the 3'-end followed by a string of poly U sequences,and the function is to terminate the gene transcription process.In addition,some studies have shown that the terminator also plays a role in post-transcription.However,at present,the definite degree of regulation of transcription and translation has not been studied specifically,and the quantitative and comparative analysis of the regulatory levels are also lacked.In addition,the particular effects of terminator sequence composition and secondary structure on their functions are still unclear.In response to the above problems,this paper analyzed preliminary structure-activity of the terminators in the mutation library by establishing a comprehensive characterization strategy of the terminator,and combining flow cytometry with high-throughput DNA and RNA sequencing,and then the following conclusions were obtained:(1)Characterized the strength of the terminator from the two levels of gene transcription and translation,and defined the three characteristic parameters of the terminator,namely the transcription shut-down degree(?),upstream mRNA protection capacity(?)and the apparent termination efficiency(?)reflecting overall effect of the terminator.Corresponding to the 3characteristic parameters,we constructed 3 terminator probe plasmids(p TK-d R,p TK-s R,and p TK)based on the dual-fluorescence reporter gene system to characterize the 12 terminators of the mini terminator library from multiple angles.Through multiple regression analysis,it was found that the transcription shut-down degree and upstream mRNA protection capacity contributed almost equally to the apparent termination efficiency.Analysis of the sequence base composition of the twelve terminators indicated that a higher proportion of GC bases in the terminator stem structure might make it a higher transcription shut-down degree.(2)Designed a terminator mutation model and constructed a terminator mutation library with a capacity of 2134 by degenerate base mutation.Using flow cytometry,terminators in the mutation library were divided into 7 subgroups according to the differences in expression intensities of upstream and downstream genes,and the relative expression intensity span of downstream fluorescent genes of the library was 5.09 times.Through the statistics of high-throughput sequencing,it was found that there was a certain inverse relationship between the upstream and downstream genes regulated by different strength terminators(R~2=0.5412).The analysis suggested that the strong terminator might increase the recycling efficiency of transcription factors such as RNA polymerase and protect the mRNA protection of upstream genes at the same time.In terms of the base composition of the terminator sequence,the key sites N13,N12,N29 and Y27 were obtained by statistical analysis of the relative frequencies of the four bases at each mutation site of the terminator sequence.The secondary structure of the terminator was statistically analyzed and the effects of the loop structure and U-tract structure of the terminator mutation library on the relative termination strength score were studied.It was found that there was a certain difference between the terminator hairpin loop and U-tract Interaction.(3)A terminator mutation library with a capacity of 2440 was obtained by RNA sequencing.According to statistics,it was speculated that the gene transcription termination site might be36 bp downstream of the terminator,and the protective effect of the terminator on mRNA hydrolysis was verified.In addition,we found that evaluating the terminator strength from the perspective of downstream gene expression would overestimate the actual level of transcription termination,and quantitative analysis from the transcription level could more accurately evaluate the terminator strength.In the structure-activity relationship of terminators,the mutation sites that had a greater influence on termination ratio were N9,N12,N20 and N29.Among them,the N12 and N29 positions were consistent with the key positions of the terminator sequence obtained by DNA sequencing analysis.In summary,this study established a comprehensive terminator characterization strategy,and constructed a terminator element library with an intensity span of 5.09 times.By analyzing the structure-function relationship of the terminator,the basic information of the terminator regulation mechanism was supplemented.It also provided guidance for rational design and application of terminators.