Most riboswitches are characterized by two components, an aptamer domain that folds into a unique ligand binding pocket to interact with the ligand, and an expression platform that converts folding changes in the apta...Most riboswitches are characterized by two components, an aptamer domain that folds into a unique ligand binding pocket to interact with the ligand, and an expression platform that converts folding changes in the aptamer into changes in gene expression. Using the recently developed systematic helix-based computational method, we theoretically studied the refolding and co-transcriptional folding behaviors of the purine riboswitch aptamers from Bacillus subtilis xpt-pbu X guanine riboswitch and Vibrio vulnificus add adenine riboswitch. Despite several intermediate structures persisting a short time during the transcription, helices P2, P3 and P1 fold in turn for both aptamers. Although some misfolded structures are observed during the refolding process, the RNAs can fold into the ligand binding pocket structure containing helices P2, P3 and P1 within a few seconds, suggesting the aptamer domains are highly evolved. The purine riboswitch aptamers can quickly fold into the ligand binding pocket structure even at a high transcription speed, possibly because formation of this structure is the necessary prerequisite for the riboswitch to bind its ligand and then regulate relevant gene expression.展开更多
Lactiplantibacillus plantarum is a metabolically versatile lactic acid bacterium found in matrices of animal and plant origin,where it contributes to fermentation.Many L.plantarum strains carry a complete riboflavin b...Lactiplantibacillus plantarum is a metabolically versatile lactic acid bacterium found in matrices of animal and plant origin,where it contributes to fermentation.Many L.plantarum strains carry a complete riboflavin biosynthesis(rib)operon that is tightly regulated by an FMN riboswitch,which contains a sensing domain(aptamer)that,upon binding to the effector(FMN),induces an ON-to-OFF conformational change in the expression platform.Riboflavin-overproducing L.plantarum strains carrying mutations in the rib operon ribos-witch have previously been selected by exposure to roseoflavin and used to produce vitamin-biofortified func-tional foods.Here,we have tested the real-time production of riboflavin by 11 mutants and found variable yields ranging from 0.9 to 6.6 mg/L.Using some of these mutants,we have developed an efficient screening method,based on imaging and quantifying the fluorescence of riboflavin in individual colonies,to identify the most effective riboflavin overproducers.Each of the 11 riboflavin-overproducing mutants had a single mutation in the aptamer of the rib operon riboswitch.To assess the riboswitch function,we have determined the transcription start site of the L.plantarum rib operon and constructed transcriptional fusions between the mCherry-encoding gene from plasmid pRCR and different riboswitch variants containing wild-type and mutant aptamers.The regulatory activity and effector responsiveness of the riboswitch variants were monitored by measuring mCherry fluorescence in L.plantarum host cells,carrying different plasmid constructs,that were cultivated with or without riboflavin.The results allowed the identification of nucleotides and secondary structures relevant for the effector-dependent regulatory activity of the riboswitch.展开更多
Riboswitches are conserved RNA elements that specifically recognize the cognate metabolites and regulate downstream gene expression involved in the metabolic pathways.To date,two classes of xanthine-responsive riboswi...Riboswitches are conserved RNA elements that specifically recognize the cognate metabolites and regulate downstream gene expression involved in the metabolic pathways.To date,two classes of xanthine-responsive riboswitches involved in xanthine homeostasis have been identified.The recently reported xanthine-II riboswitch originates from guanine riboswitch family,featuring a single U-to-G mutation and several nucleotide insertions.Here,we report the complex structure of xanthine-II riboswitch bound to xanthine.The tertiary structure of xanthine-II riboswitch adopts a three-way junction scaffold similar to that of guanine riboswitch.However,the distinctive mutation and insertions in xanthine-II riboswitch facilitate the formation of a highly specific binding pocket for xanthine,distinguishing it from guanine riboswitches.Xanthine is bound in the junction region,forming a base triple with C64 and the mutant nucleotide G37,and is sandwiched by one base pair U8-A38 and one base triple A7-C36-U65.Structural alignment and ligand recognition specificity of the xanthine-II riboswitch are further verified by ligand-binding assays of structure-based mutation using isothermal titration calorimetry.Furthermore,leveraging the ligand specificity of the xanthine-II riboswitch,we develop a highly specific and sensitive biosensor for xanthine detection by fusing xanthine-II riboswitch with Pepper fluorogenic aptamer,highlighting the potential applications of xanthine-II riboswitch in diagnosing diseases related to xanthine metabolism disorders.展开更多
Riboswitches are highly conserved RNA elements that located in the 5’-UTR of m RNAs,which undergo real-time structure conformational change to achieve the regulation of downstream gene expression by sensing their cog...Riboswitches are highly conserved RNA elements that located in the 5’-UTR of m RNAs,which undergo real-time structure conformational change to achieve the regulation of downstream gene expression by sensing their cognate ligands.S-adenosylmethionine(SAM)is a ubiquitous methyl donor for transmethylation reactions in all living organisms.SAM riboswitch is one of the most abundant riboswitches that bind to SAM with high affinity and selectivity,serving as regulatory modules in multiple metabolic pathways.To date,seven SAM-specific riboswitch classes that belong to four families,one SAM/SAH riboswitch and one SAH riboswitch have been identified.Each SAM riboswitch family has a well-organized tertiary core scaffold to support their unique ligand-specific binding pocket.In this review,we summarize the current research progress on the distribution,structure,ligand recognition and gene regulation mechanism of these SAM-related riboswitch families,and further discuss their evolutionary prospects and potential applications.展开更多
Riboswitches are functional RNA elements that regulate gene expression by directly detecting metabolites.Twenty years have passed since it was first discovered,researches on riboswitches are becoming increasingly stan...Riboswitches are functional RNA elements that regulate gene expression by directly detecting metabolites.Twenty years have passed since it was first discovered,researches on riboswitches are becoming increasingly standardized and refined,which could significantly promote people’s cognition of RNA function as well.Here,we focus on some representative orphan riboswitches,enumerate the structural and functional transformation and artificial design of riboswitches including the coupling with ribozymes,hoping to attain a comprehensive understanding of riboswitch research.展开更多
目的建立氟化物核糖开关转录反应体系以及成簇的规律间隔的短回文重复序列/Cas12a蛋白(clustered regularly interspaced short palindromic repeats-associated protein 12a,CRISPR/Cas12a)检测氟含量的方法。方法本研究利用氟化物核...目的建立氟化物核糖开关转录反应体系以及成簇的规律间隔的短回文重复序列/Cas12a蛋白(clustered regularly interspaced short palindromic repeats-associated protein 12a,CRISPR/Cas12a)检测氟含量的方法。方法本研究利用氟化物核糖开关在有氟或无氟下转录全长或非全长RNA并与CRISPR/Cas12a系统相结合,通过荧光法读取实验结果。对优化的CRISPR/Cas12a检测体系进行选择性和干扰性分析实验,并用于南极磷虾氟含量的检测。结果本研究成功建立了基于氟化物核糖开关转录反应体系CRISPR/Cas12a荧光检测方法,可在1.5 h内完成对样品的检测,检测结果与国家标准方法氟离子选择电极法基本保持一致。结论本研究建立的基于氟化物核糖开关转录反应和CRISPR/Cas12a的南极磷虾中氟含量检测方法具有高特异性和准确性,在氟含量的检测方面具有应用价值。展开更多
Transcription attenuation in response to the availability of a specific amino acid is believed to be controlled by alternative configurations of RNA secondary structures that lead to the arrest of translation or the r...Transcription attenuation in response to the availability of a specific amino acid is believed to be controlled by alternative configurations of RNA secondary structures that lead to the arrest of translation or the release of the arrested ribosome from the leader mRNA molecule.In this study,we first report a possible example of the DnaA‐dependent riboswitch for transcription attenuation in Escherichia coli.We show that(i)DnaA regulates the transcription of the structural genes but not that of the leader hisL gene;(ii)DnaA might bind to rDnaA boxes present in the HisL‐SL RNA,and subsequently attenuate the transcription of the operon;(iii)the HisL‐SL RNA and rDnaA boxes are phylogenetically conserved and evolutionarily important;and(iv)the translating ribosome is required for deattenuation of the his operon,whereas tRNA^(His) strengthens attenuation.This mechanism seems to be phylogenetically conserved in Gram‐negative bacteria and evolutionarily important.展开更多
With the support by the National Natural Science Foundation of China,the research team led by Dr.Liu Yuchen(刘宇辰)at the State Engineering Laboratory of Medical Key Technologies Application of Synthetic Biology,Shenz...With the support by the National Natural Science Foundation of China,the research team led by Dr.Liu Yuchen(刘宇辰)at the State Engineering Laboratory of Medical Key Technologies Application of Synthetic Biology,Shenzhen Second People’s Hospital,the First Affiliated Hospital of Shenzhen University,developed a type of CRISPR-based riboswitch,which was published in Nature Methods(2016,doi:10.展开更多
基金Supported by the National Natural Science Foundation of China(31600592)Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive Utilization,Hubei Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains(2017BX08)
文摘Most riboswitches are characterized by two components, an aptamer domain that folds into a unique ligand binding pocket to interact with the ligand, and an expression platform that converts folding changes in the aptamer into changes in gene expression. Using the recently developed systematic helix-based computational method, we theoretically studied the refolding and co-transcriptional folding behaviors of the purine riboswitch aptamers from Bacillus subtilis xpt-pbu X guanine riboswitch and Vibrio vulnificus add adenine riboswitch. Despite several intermediate structures persisting a short time during the transcription, helices P2, P3 and P1 fold in turn for both aptamers. Although some misfolded structures are observed during the refolding process, the RNAs can fold into the ligand binding pocket structure containing helices P2, P3 and P1 within a few seconds, suggesting the aptamer domains are highly evolved. The purine riboswitch aptamers can quickly fold into the ligand binding pocket structure even at a high transcription speed, possibly because formation of this structure is the necessary prerequisite for the riboswitch to bind its ligand and then regulate relevant gene expression.
基金supported by the Spanish Ministry of Science,Innovation and Universities(grant number RT12018-097114-B-I00)the Consejo Superior de Investigaciones Científicas(grant number 2022AEP028)supported by the funding of the European Union Next Generation EU[PNRR—Mission 4 Component 2,Investment 1.4—D.D.1032 June 17,2022,CN00000022]within the Agritech National Research Centre for Agricultural Technologies.
文摘Lactiplantibacillus plantarum is a metabolically versatile lactic acid bacterium found in matrices of animal and plant origin,where it contributes to fermentation.Many L.plantarum strains carry a complete riboflavin biosynthesis(rib)operon that is tightly regulated by an FMN riboswitch,which contains a sensing domain(aptamer)that,upon binding to the effector(FMN),induces an ON-to-OFF conformational change in the expression platform.Riboflavin-overproducing L.plantarum strains carrying mutations in the rib operon ribos-witch have previously been selected by exposure to roseoflavin and used to produce vitamin-biofortified func-tional foods.Here,we have tested the real-time production of riboflavin by 11 mutants and found variable yields ranging from 0.9 to 6.6 mg/L.Using some of these mutants,we have developed an efficient screening method,based on imaging and quantifying the fluorescence of riboflavin in individual colonies,to identify the most effective riboflavin overproducers.Each of the 11 riboflavin-overproducing mutants had a single mutation in the aptamer of the rib operon riboswitch.To assess the riboswitch function,we have determined the transcription start site of the L.plantarum rib operon and constructed transcriptional fusions between the mCherry-encoding gene from plasmid pRCR and different riboswitch variants containing wild-type and mutant aptamers.The regulatory activity and effector responsiveness of the riboswitch variants were monitored by measuring mCherry fluorescence in L.plantarum host cells,carrying different plasmid constructs,that were cultivated with or without riboflavin.The results allowed the identification of nucleotides and secondary structures relevant for the effector-dependent regulatory activity of the riboswitch.
基金supported by the National Key Research and Development Project of China (2021YFC2300300,2023YFC2604300)the National Natural Science Foundation of China (32325029,91940302,91640104)。
文摘Riboswitches are conserved RNA elements that specifically recognize the cognate metabolites and regulate downstream gene expression involved in the metabolic pathways.To date,two classes of xanthine-responsive riboswitches involved in xanthine homeostasis have been identified.The recently reported xanthine-II riboswitch originates from guanine riboswitch family,featuring a single U-to-G mutation and several nucleotide insertions.Here,we report the complex structure of xanthine-II riboswitch bound to xanthine.The tertiary structure of xanthine-II riboswitch adopts a three-way junction scaffold similar to that of guanine riboswitch.However,the distinctive mutation and insertions in xanthine-II riboswitch facilitate the formation of a highly specific binding pocket for xanthine,distinguishing it from guanine riboswitches.Xanthine is bound in the junction region,forming a base triple with C64 and the mutant nucleotide G37,and is sandwiched by one base pair U8-A38 and one base triple A7-C36-U65.Structural alignment and ligand recognition specificity of the xanthine-II riboswitch are further verified by ligand-binding assays of structure-based mutation using isothermal titration calorimetry.Furthermore,leveraging the ligand specificity of the xanthine-II riboswitch,we develop a highly specific and sensitive biosensor for xanthine detection by fusing xanthine-II riboswitch with Pepper fluorogenic aptamer,highlighting the potential applications of xanthine-II riboswitch in diagnosing diseases related to xanthine metabolism disorders.
基金supported by the National Natural Science Foundation of China(32022039,31870810,91940302,91640104)the National Key Research and Development Project of China(2021YFC2300300)+2 种基金the China Postdoctoral Science Foundation(2022M713637)the Outstanding Youth Fund of Zhejiang Province(LR19C050003)the Fundamental Research Funds for the Central Universities(2017QN81010)。
文摘Riboswitches are highly conserved RNA elements that located in the 5’-UTR of m RNAs,which undergo real-time structure conformational change to achieve the regulation of downstream gene expression by sensing their cognate ligands.S-adenosylmethionine(SAM)is a ubiquitous methyl donor for transmethylation reactions in all living organisms.SAM riboswitch is one of the most abundant riboswitches that bind to SAM with high affinity and selectivity,serving as regulatory modules in multiple metabolic pathways.To date,seven SAM-specific riboswitch classes that belong to four families,one SAM/SAH riboswitch and one SAH riboswitch have been identified.Each SAM riboswitch family has a well-organized tertiary core scaffold to support their unique ligand-specific binding pocket.In this review,we summarize the current research progress on the distribution,structure,ligand recognition and gene regulation mechanism of these SAM-related riboswitch families,and further discuss their evolutionary prospects and potential applications.
基金the National Key Research and Development Program of China(2021YFC2100700)the National Natural Science Foundation of China(Grant NSFC-22278313).
文摘Riboswitches are functional RNA elements that regulate gene expression by directly detecting metabolites.Twenty years have passed since it was first discovered,researches on riboswitches are becoming increasingly standardized and refined,which could significantly promote people’s cognition of RNA function as well.Here,we focus on some representative orphan riboswitches,enumerate the structural and functional transformation and artificial design of riboswitches including the coupling with ribozymes,hoping to attain a comprehensive understanding of riboswitch research.
文摘目的建立氟化物核糖开关转录反应体系以及成簇的规律间隔的短回文重复序列/Cas12a蛋白(clustered regularly interspaced short palindromic repeats-associated protein 12a,CRISPR/Cas12a)检测氟含量的方法。方法本研究利用氟化物核糖开关在有氟或无氟下转录全长或非全长RNA并与CRISPR/Cas12a系统相结合,通过荧光法读取实验结果。对优化的CRISPR/Cas12a检测体系进行选择性和干扰性分析实验,并用于南极磷虾氟含量的检测。结果本研究成功建立了基于氟化物核糖开关转录反应体系CRISPR/Cas12a荧光检测方法,可在1.5 h内完成对样品的检测,检测结果与国家标准方法氟离子选择电极法基本保持一致。结论本研究建立的基于氟化物核糖开关转录反应和CRISPR/Cas12a的南极磷虾中氟含量检测方法具有高特异性和准确性,在氟含量的检测方面具有应用价值。
基金supported by grants from the National Natural Science Foundation of China(Grant no.32260233 to Morigen)the Science and Technology Foundation of Inner Mongolia(Inner Mongolia Key Laboratory for Molecular Regulation of the Cell,Grant no.2021PT0002).
文摘Transcription attenuation in response to the availability of a specific amino acid is believed to be controlled by alternative configurations of RNA secondary structures that lead to the arrest of translation or the release of the arrested ribosome from the leader mRNA molecule.In this study,we first report a possible example of the DnaA‐dependent riboswitch for transcription attenuation in Escherichia coli.We show that(i)DnaA regulates the transcription of the structural genes but not that of the leader hisL gene;(ii)DnaA might bind to rDnaA boxes present in the HisL‐SL RNA,and subsequently attenuate the transcription of the operon;(iii)the HisL‐SL RNA and rDnaA boxes are phylogenetically conserved and evolutionarily important;and(iv)the translating ribosome is required for deattenuation of the his operon,whereas tRNA^(His) strengthens attenuation.This mechanism seems to be phylogenetically conserved in Gram‐negative bacteria and evolutionarily important.
文摘With the support by the National Natural Science Foundation of China,the research team led by Dr.Liu Yuchen(刘宇辰)at the State Engineering Laboratory of Medical Key Technologies Application of Synthetic Biology,Shenzhen Second People’s Hospital,the First Affiliated Hospital of Shenzhen University,developed a type of CRISPR-based riboswitch,which was published in Nature Methods(2016,doi:10.
