The Construction Of The Terminator Library And Analysis Of The Structure-activity Relationship In E.coli

The intrinsic terminator,also known as theρfactor-independent terminator,relies on the secondary structure formed by the folding of its own RNA sequence to terminate the process of gene transcription,which is one of the necessary elements for the construction of gene expression cassettes.The secondary structure of the intrinsic terminator mainly consists of three parts:a hairpin structure consisting of a 5-9 nt GC-rich stem and a 3-5 nt loop,a A-tract and U-tract structure located at upstream and downstream of the hairpin structure respectively.Instrinic terminators are widely used in gene expression regulation because of their simple mechanism and high termination efficiency.In eukaryotes,a large number of studies have confirmed that the existence of terminators can effectively prevent upstream transcripts from being degraded and increase the expression level of transcription.However,in prokaryotes,there is still a lack of targeted research on the effect of terminators on the expression of upstream genes.Moreover,different strengths of terminators play different roles in the regulation of gene expression.In addition to using experimental methods to characterize the terminator strength,the strategy of preliminarily classifying the terminator strength by computational methods also has important research significance.In order to solve the problem,a comprehensive and systematic study of the structure-activity relationship of terminators from both transcriptional and translational levels are needed to conduct.Based on the above problems:this paper firstly established a small sequence library to characterize the effect of insertion with and without terminator characteristic sequences on the expression of upstream and downstream genes.Secondly,based on Flow Seq technology,the structure-activity relationship of terminators with different phenotypes were analyzed from the perspective of sequence and thermodynamics.Finally,terminator libraries were established,with the help of RNA-Seq technology,the termination sites of the terminator were counted at the transcriptional level.Meanwhile,the adaptability between different promoters and terminators was further explored.The conclusions are as follow:(1)Intrinsic terminators can significantly inhibit the expression of downstream genes,and the sequence with hairpin structure at the 3’end may increase the expression of upstream genes.A total of 19 recombinant plasmids with and without terminator characteristic sequences were constructed,by means of dual fluorescent probe plasmids and flow cytometry,the effects of the two sets of sequence with different characteristics on the expression of upstream and downstream reporter genes were compared at the single cell level.The results showed that the expression of downstream genes in the terminator sequence group was significantly inhibited.At the same time,the expression of upstream genes within the group displayed a large difference whether it was the terminator group or the spacer sequence group,indicating that the sequence at the 3’end of the coding gene would have a certain impact on the expression of the upstream gene.However,with the decrease of the average free energy per base of the inserted sequence,the expression intensity of the upstream gene showed an upward trend,indicating that in the 3’end of the coding gene,the sequence that is more likely to form a hairpin structure may have a certain protective effect on the upstream gene.Further characterization of the transcription levels of the reporter genes that located at downstream of the strong,medium and weak terminators spy,phe A and rec A confirmed that the timely termination of transcription by the terminator was the main reason for the decline in the expression of translation levels.(2)The structure-activity relationship analysis of intrinsic terminators in E.coli based on Flow Seq technology.The phenotype and genotype information of 808 terminators were collected by flow cytometry sorting combined with high-throughput sequencing.The analysis of the upstream and downstream fluorescent gene expression of 225 high-quality terminator sequences showed that,when the expression of downstream gene enhanced,the expression of upstream genes showed a downward trend(r=-0.6),indicating that the timely termination of the transcription process is beneficial to the improvement of the efficiency of RNA polymerase cycle and the growth of cells.The structure-activity relationship analysis of 214 strong and weak terminators with preference in each bin showed that the terminator with longer stem length and more perfect U-tract structure,namely the terminator with low free energy of the hairpin structure(ΔG_H)and the loop structure(ΔG_L),a high free energy in U-tract strcture(ΔG_U)was more likely to be strong terminator.Based on the XGBoost algorithm,a classification model of terminator strength was further established and the experimental verification results used by 20 synthetic terminators showed that the model can distinguish strong and weak terminators to a certain degree.(3)The construction of terminator mutation library and determination of termination site based on second generation sequencing technology.The terminator thr L was used as a mutation template,mutation sites were designed at the three positions of the terminator including loop structure,stem structure,and A-tract/U-tract structure.The second-strand c DNA was amplified by klenow enzyme and the terminator mutant fragment was connected to the double fluorescent probe plasmid to complete the construction of the terminator library.The results of RNA-Seq showed that the sequences in the three mutant libraries all had an interruption peak at the downstream 47 bp from the terminator,and after this site,the terminator sequences in the libraries appeared to terminate the transcription one after another.The results of the adaptability of promoters and terminators showed that when the promoter strength was strong,the phenomenon of leaky expression was serious,however,the matching strong terminator can effectively prevent the situation,thereby improving the utilization efficiency of cells.In conclusion,this study compared the effect of terminator sequence and non-terminator sequence on gene expression,determined the significant inhibitory effect of terminator on downstream genes,and found that the 3’-end sequence with the hairpin structure increased the expression of upstream genes;based on Flow Seq technicology,the structure-activity relationship of the terminator was analysized and a clasification model of terminator strength was established;three terminator mutation libraries at different positions were established,the termination positions of sequences in each library were counted and the differences between different promoters and terminators were analyzed.The study provided guidance for rational design of terminator-related functional elements.