Riboswitch Regulation ofGene Expression Riboswitches are present in untranslated regions of mRNAs. Their purpose is to regulate gene expression in response to binding small molecule metabolites.Riboswitches are defined by two main criteria:•Direct (protein-free) binding of metabolite to RNA•Metabolite-dependent regulation of genesThis movie demonstrates the molecular events common to most bacterial riboswitches. Resources are listed at the end of the movie. Part I:Gene Regulation by aTypical Riboswitch Bacterial riboswitches are present in the 5´ untranslated region of mRNAs.Transcription is regulated by the gene promoter and transcription initiation factors … RNA polymerase initiates transcription.The RNA folds intramolecularly in local regions of complementarity, presumably, while transcription is proceeding.A long untranslated leader is produced first.Nascent RNARNApolymeraseDNA template RNApolymeraseCase 1:Cellular concentration of metabolite is too low to occupy the riboswitch binding site.Transcription and …3421RNApolymerase UUUUUAUGRNApolymeraseCase 1:Cellular concentration of metabolite is too low to occupy the riboswitch binding site.Transcription and intramolecular RNA folding continue.34213421 UUUUUAUGCase 1:Cellular concentration of metabolite is too low to occupy the riboswitch binding site.Translation is initiated.RibosomeTypically the new mRNA codes for a biosynthetic or transport protein that raises the intracellular level of the metabolite. Gene regulation (next case) is accomplished by variations in the interactions of the regions highlighted in orange. Transcription and intramolecular RNA folding continue.3421 Case 2:Cellular concentration of metabolite (X) is high.Intramolecular folding can lead to an alternate conformation.RNA polymerase produces the long untranslated leader region.The alternate riboswitch conformation is stable when metabolite is bound. XXXXXRNApolymeraseXNascent RNADNA template Case 2:Cellular concentration of metabolite (X) is high.Intramolecular folding can lead to an alternate conformation.RNA polymerase produces the long untranslated leader region.The alternate riboswitch conformation is stable when metabolite is bound. XXXXXXTranscription continues.UUUUURNApolymerase3421 Case 2:Cellular concentration of metabolite (X) is high.XXXXXTranscription continues.RNApolymeraseNow, RNA folding leads to formation of an intrinsic terminator.UUUUUXX34213421 Case 2:Cellular concentration of metabolite (X) is high.XXXXXTranscription continues.RNApolymeraseNow, RNA folding leads to formation of an intrinsic terminator.UUUUUXThe transcript is never completed and the metabolite biosynthetic or transport protein is not produced.3421 ReviewCase 1:Metabolite is limited.Case 2:Metabolite is abundant.UUUUUXUUUUUAUGORFTranscription is completed.Biosynthetic and/or transport proteins are expressed.Transcription is terminated.Proteins are downregulated.XXXX34213421 Part II:The Expanding Universeof Riboswitches … an ‘expression platform’ for gene regulation.A metabolite-binding‘aptamer’ domain and …Riboswitch FunctionsUUUUUX3421Riboswitches were defined earlier by two main criteria:•Direct (protein-free) binding of metabolite to RNA•Metabolite-dependent regulation of genesThese two activities are accomplished by two functionally separate domains on the RNA: … an ‘expression platform’ for gene regulation.A metabolite-binding‘aptamer’ domain and …Riboswitch FunctionsUUUUUX3421•Direct (protein-free) binding of metabolite to RNA•Metabolite-dependent regulation of genesThese two activities are accomplished by two functionally separate domains on the RNA:Because of the modular nature of RNA structures, different types of expression platform can be linked to the conserved aptamer domain.This leads to variations in riboswitch mechanism … Riboswitch MechanismsThis movie showed the most common case for bacterial riboswitches:Ligand binding leads totranscription termination and reduced gene expression.UUUUUX5´ORFX Riboswitch MechanismsRiboswitches also regulate translation and anti-termination …Ligand binding leads totranscription termination and reduced gene expression.UUUUUX5´ORFLigand binding sequestersthe Shine-Dalgarno sequence and reduces gene expression.Ligand binding leads toantiterminator formation and increased gene expression.UUUUUX5´AUGORFXXX5´AUGORFAGGAGGX Riboswitch MechanismsThey also affect RNA integrity, and perhaps splicing and stability.Ligand binding leads tomRNA cleavage by a new natural ribozyme.X5´Ligand binding could controlsplicing in eukayotes.Possibly, ligand binding to the 3´ untranslated region could affect mRNA stability.AUGORFX5´AUGORFAGGUACGGAAAAAX5´AUGORFX Riboswitch LigandsFigure shows the chemical structures of riboswitch ligands and schematics of conserved secondary structure in riboswitches.There are 8 confirmed riboswitches with unique metabolite ligands.Many more conserved RNA motifs are currently under investigation. Riboswitch Gene RegulationRiboswitches are an important mechanism of gene regulation. For example, nearly 2% of the genes of the model organism Bacillus subtilis appear to be controlled by riboswitches. Metabolite RecognitiontRNARiboswitch11-mer TRAP complexCoenzymeB12Average metaboliteRelative sizes of some molecules recognized for gene regulation (metabolites and tRNAs) and some agents that recognize them (riboswitches and TRAP complex).Riboswitches are an economical way to see small molecules 核糖开关• 是一类位于mRNA 3’或5’-UTR上的能够结合小分子代谢物以调控基因的转录和翻译的mRNA元件• 它与小分子代谢物的结合不依赖于任何蛋白质，从而使构象发生改变，在转录或翻译水平调控基因的表达。
•到目前为至已经发现了不少于12种核糖开关：• 维生素B12核糖开关• 硫胺素焦磷酸（ TPP）核糖开关• FMN（黄素单核苷酸）核糖开关• SAM（S-甲腺甲硫氨酸）核糖开关• 赖氨酸核糖开关• 鸟嘌呤核糖开关• 腺嘌呤核糖开关• GlmS核糖开关（调控葡糖胺-6-磷酸合成）• 甘氨酸核糖开关• mgtA核糖开关（mgtA为沙门氏菌Mg2+转运载体）• SAH核糖开关(调控S-腺苷同型半胱氨酸) • preQ1核糖开关(调控Q核苷前体7-甲氨基-7去氮鸟嘌呤（7-aminomethyl-7-deazaguanine）合成) •核糖开关如果按作用类型可分为• 抑制型（即底物存在时抑制基因表达）• 激活型（当底物存在时基因开始表达）• • 其中已知的绝大部分核糖开关都是抑制型，只有腺嘌呤核糖开关是激活型 •如按存在位置核糖开关可分为:• 3’UTR核糖开关• 5’UTR核糖开关• 到目前为止只有TPP核糖开关在不同物种中存在的位置不一样，TPP核糖开关在高等植物中位于3’UTR，而在细菌、真菌和绿藻中则位于5’UTR。
其它的核糖开关均位于5’UTR •核糖开关的结构特点：• 核糖开关主要由适体结构域（aptamer domain, AD）和表达结构域(expression domain, EPD)组成；• AD序列保守，不需要蛋白质的参与，直接与小分子（即适体，aptamer）代谢物结合，AD在核糖开关中主要起RNA传感器的作用• EPD序列不保守，是核糖开关的表达模块，它的主要功能是在AD与适体结合后，引起AD构象变化后迅速作出反应，导致EPD构象变化，通过变构效应调控基因表达 核糖开关的一般作用机制核糖开关的一般作用机制• 不同的核糖开关有不同的作用机制目前普遍认为核糖开关主要在转录和翻译两个水平上对基因表达进行调控，分为抑制和激活两类• 适体与AD特异性结合，使EPD构象发生变化，形成有选择性的茎环结构，导致mRNA转录提前结束或者抑制翻译的起始 •转录水平的调控：• 在mRNA末端存在“抗抗终止子”、“抗终止子”、“终止子”• 当AD未与适体结合时，抗终止子与终止子结合，使转录顺利进行；• 当AD与适体结合后，导致mRNA构象发生变化，抗抗终止子与抗终止子结合，从而终止子形成发夹结构，转录终止。
•翻译水平的调控：• 在起始密码上游，存在“抗抗SD”，“抗SD”，“SD”序列• 当AD未与适体结合时，抗抗SD与抗SD结合，使翻译顺利进行；• 当AD与适体结合后，使抗SD与SD结合，导致翻译不能进行 腺嘌呤核糖开关的作用机制• 腺嘌呤核糖开关的AD主要由三个螺旋（P1，P2，P3）组成，他们的形状好像一个倒置的“h”，P1与P3同轴，P1在下，P3在上，P2在P3的左侧，腺嘌呤结合位点在三个螺旋的结合处，其中P2和P3存在着两个的G-C 碱基对（G37与C61，G38与C60），它们在AD空间构象发生改变时，起着十分重要的作用 • Lemay等认为AD存在两种构象，即“U”和“F”；• 当腺嘌呤不存在时，P3上的两个的G-C 碱基对与P2之间的氢键发生断裂，导致P2与P3一端分开，P1、P2、P3之间较为分散，此时的构象称之为“U”；• 当腺嘌呤存在时，P2与P3之间又形成了氢键并结合在一起，从而P1、P2和P3折叠在一起，准备与腺嘌呤结合，即为“F” TPP核糖开关的作用机制 • TPP核糖开关主要由五个螺旋（P1，P2，P3，P4，P5）组成一个倒置的“h”型;• 其中P1，P2，P3同轴，P3在上，P2居中，P1在下。
P4，P5同轴，P5在上，P4在下• P1，P2，P3与TPP的嘧啶部分连接，P4，P5与TPP的焦磷酸盐部分连接 • TPP的嘧啶环的连接部位在P3与P2结合部J3/2；在J3/2存在一段十分保守的序列UGAGA；其中U39与A43能够形成U-A碱基对，这就使得G40，A41和G42能够形成一段弧，有利于与TPP的嘧啶环结合，TPP的嘧啶通过氢键与G42连接在一起• TPP的焦磷酸盐部分通过二价的金属离子与P4，P5的结合部位J4/5中的G78连接在一起 • 当外界环境中的TPP浓度低于TPP核糖开关的亲和力时，TPP核糖开关不能与TPP结合，从而使TPP的合成顺利进行• 当TPP浓度达到TPP核糖开关的亲和力时，TPP核糖开关便与TPP结合，这时TPP核糖开关的AD空间构象发生改变，形成有选择性的茎环状结构导致转录提前终止，或者阻止16S rRNA与SD序列的结合从而破坏了翻译的起始 核糖开关的应用核糖开关的应用•药物设计：• 核糖开关具有高度的保守性，尤其是它的AD结构域保守性更强，这就使得它可以作为新药的作用靶点，使其用于基因治疗成为可能；• 将新药设计成与对应的核糖开关的适体具有类似结构，使药物能够与核糖开关的AD结构域结合，改变核糖开关的构象，形成有选择性的茎环结构，使该基因的转录终止或者翻译提前结束，来抑制基因的表达，从而达到基因治疗的目的。
•新基因的功能：• 将核糖开关引入到新基因的5’UTR或3’UTR，通过该基因的表达与否进而研究新基因的功能 •植物生物传感器• 由于核糖开关对于适体具有严格的特异性和不需要蛋白质的参与,使其在植物生物传感器研制方面也具有独特优势• Bocobza等已成功地将TPP核糖开关的适体结构域连接到植物黄色荧光蛋白报告基因3’UTR中去当TPP的浓度低于100nM时，该植物的叶片有很亮的黄色荧光，当TPP的浓度位于100nM和1μM时荧光明显变暗，当TPP的浓度大于1μM基本不发荧光这为核糖开关在生物传感器方面的应用提供了重要的指导。