The intrinsic terminator in prokaryotic forms secondary RNA structure and terminates the transcription.However,leaking transcription is common due to varied terminator strength.Besides of the representative hairpin an...The intrinsic terminator in prokaryotic forms secondary RNA structure and terminates the transcription.However,leaking transcription is common due to varied terminator strength.Besides of the representative hairpin and U-tract structure,detailed sequence and thermodynamic features of terminators were not completely clear,and the effect of terminator on the upstream gene expression was unclearly.Thus,it is still challenging to use terminator to control expression with higher precision.Here,in E.Coli,we firstly determined the effect of the 3′-end sequences including spacer sequences and terminator sequences on the expression of upstream and downstream genes.Secondly,terminator mutation library was constructed,and the thermodynamic and sequence features differing in the termination efficiency were analyzed using the FlowSeq technique.The result showed that under the regulation of terminators,a negative correlation was presented between the expression of upstream and downstream genes(r=0.60),and the terminators with lower free energy corelated with higher upstream gene expression.Meanwhile,the terminator with longer stem length,more compact loop and perfect U-tract structure was benefit to the transcription termination.Finally,a terminator strength classification model was established,and the verification experiment based on 20 synthetic terminators indicated that the model can distinguish strong and weak terminators to certain extent.The results help to elucidate the role of terminators in gene expression,and the key factors identified are crucial for rational design of terminators,and the model provided a method for terminator strength prediction.展开更多
The ribosome-binding site(RBS)in the 5′untranslated region is recognized by 16S rRNA to start translation and is an essential element of the gene expression system.RBSs have been widely applied in regulating gene exp...The ribosome-binding site(RBS)in the 5′untranslated region is recognized by 16S rRNA to start translation and is an essential element of the gene expression system.RBSs have been widely applied in regulating gene expression in various scenarios,including Gram-negative or Gram-positive bacteria.Here,we first rationally designed and constructed an RBS mutant library containing 66 RBSs.The strength of these RBSs in E.coli and C.glutamicum was characterized individually.The RBS strength spanned about 200 and 15 times in the two species,respectively.The strength of RBSs in C.glutamicum was generally lower than that of in E.coli.A total of 18 RBSs showed similar strength(within twofold differences)between the species in our study,and the correlation analysis of the strength of RBSs between E.coli and C.glutamicum(R^(2)=0.7483)revealed that these RBSs can be used across species.The sequence analysis revealed that the RBS region with two Ts stated was beneficial for RBS to function cross-species.The RBS characterized here can be used to precisely regulate gene expression in both hosts,and the characteristics of cross-species RBSs provide basic information for RBS rational design.展开更多
基金This study was supported by the National Key Research and Development Program of China[2018YFA0900300]National Natural Science Foundation of China[32171421].
文摘The intrinsic terminator in prokaryotic forms secondary RNA structure and terminates the transcription.However,leaking transcription is common due to varied terminator strength.Besides of the representative hairpin and U-tract structure,detailed sequence and thermodynamic features of terminators were not completely clear,and the effect of terminator on the upstream gene expression was unclearly.Thus,it is still challenging to use terminator to control expression with higher precision.Here,in E.Coli,we firstly determined the effect of the 3′-end sequences including spacer sequences and terminator sequences on the expression of upstream and downstream genes.Secondly,terminator mutation library was constructed,and the thermodynamic and sequence features differing in the termination efficiency were analyzed using the FlowSeq technique.The result showed that under the regulation of terminators,a negative correlation was presented between the expression of upstream and downstream genes(r=0.60),and the terminators with lower free energy corelated with higher upstream gene expression.Meanwhile,the terminator with longer stem length,more compact loop and perfect U-tract structure was benefit to the transcription termination.Finally,a terminator strength classification model was established,and the verification experiment based on 20 synthetic terminators indicated that the model can distinguish strong and weak terminators to certain extent.The results help to elucidate the role of terminators in gene expression,and the key factors identified are crucial for rational design of terminators,and the model provided a method for terminator strength prediction.
基金This work was financially supported by the National Key Research and Development Program of China(2018YFA0900300)the National Natural science foundation of China(32171421).
文摘The ribosome-binding site(RBS)in the 5′untranslated region is recognized by 16S rRNA to start translation and is an essential element of the gene expression system.RBSs have been widely applied in regulating gene expression in various scenarios,including Gram-negative or Gram-positive bacteria.Here,we first rationally designed and constructed an RBS mutant library containing 66 RBSs.The strength of these RBSs in E.coli and C.glutamicum was characterized individually.The RBS strength spanned about 200 and 15 times in the two species,respectively.The strength of RBSs in C.glutamicum was generally lower than that of in E.coli.A total of 18 RBSs showed similar strength(within twofold differences)between the species in our study,and the correlation analysis of the strength of RBSs between E.coli and C.glutamicum(R^(2)=0.7483)revealed that these RBSs can be used across species.The sequence analysis revealed that the RBS region with two Ts stated was beneficial for RBS to function cross-species.The RBS characterized here can be used to precisely regulate gene expression in both hosts,and the characteristics of cross-species RBSs provide basic information for RBS rational design.
