转录和转录组学transcriptome课件.ppt

1、转录和转录组学Thanscription and TranscriptomicsReplicationReplication TranscriptionReverse Transcription Translation中心法则中心法则（the central dogma）DNARNAProteinThe Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry for 2006 toRoger D.KornbergStanford University,CA,USAfor his st

2、udies of the molecular basis of eukaryotic transcription.Roger D.Kornberg,born 1947(59)in St Louis,MO,USA(US citizen).PhD from Stanford University,CA,USA.Mrs.George A.Winzer Professor in Medicine at Stanford University School of Medicine,CA,USA.Original scientific articlesCramer,P.,Bushnell,D.A.and

3、Kornberg,R.D.(2001)Structural basis of transcription:RNA polymerase II at 2.8 ngstrom resolution.Science 292,1863-1876.Gnatt,A.L.,Cramer,P.,Fu,J.,Bushnell,D.A.and Kornberg,R.D.(2001)Structural basis of transcription:An RNA polymerase II elongation complex at 3.3 resolution.Science 292,1876-1882.Bush

4、nell,D.A.,Westover,K.D.,Davis,R.E.and Kornberg,R.D.(2004)Structural basis of transcription:An RNA polymerase II TFIIB cocrystal at 4.5 angstroms.Science 303,983-988.Review articleBoeger,H.,Bushnell,D.A.,Davis,R.,Griesenbeck,J.,Lorch,Y.,Strattan,J.S.,Westover,K.D.and Kornberg,R.D.(2005).Structural ba

5、sis of eukaryotic gene transcription.FEBS Lett.579,899-903.Severo OchoaArthur Kornberg 1/2 of the prize 1/2 of the prizeUSAUSANew York University,College of Medicine New York,NY,USAStanford University Stanford,CA,USAb.1905(in Luarca,Spain)d.1993 b.1918 The Nobel Prize in Physiology or Medicine 1959f

6、or their discovery of the mechanisms in the biological synthesis of ribonucleic acid and deoxyribonucleic acid2006年诺贝氏生理医学奖得主 Dr.Fire A and Dr Mello C核苷酸链断裂点核苷酸链断裂点槌头状结构，最简单的核酶槌头状结构，最简单的核酶 模板链模板链（template strand）也称反意义链（antisense strand)链或Watson链，即指导转录生成指导转录生成RNA的的 一股一股DNA单单链。链。5 GCAGTACATGTC.3 3 c g

7、 t c a t g t a c a g.5 5 GCAGUACAUGUC.3 N Ala.Val.His.ValC DNARNA肽肽转录转录翻译翻译编码链编码链模板链模板链535355不对称转录不对称转录 原核生物的原核生物的RNA聚合酶全酶及其在转录起聚合酶全酶及其在转录起始区的结合始区的结合DNA双链以打开，双链以打开，因子尚未脱落因子尚未脱落-35TTGACATATAAT +20 +1RNA pol保护区保护区结构基因结构基因终止点终止点+11.4.1.1.4.1.转录起始转录起始原核生物的原核生物的RNA聚合酶全酶及其在转录起始区的结合聚合酶全酶及其在转录起始区的结合DNA双链以打开

8、，双链以打开，因子尚未脱落因子尚未脱落-35TTGACATATAAT +20 +1 5 5 富富Cpolymerasepolymerase+1增强子（+）静息子（-）基础表达（启动子）结构基因-25-702.2.3.真核生物转录的特点：真核生物转录的特点：TFD TFA TFB pol/TFF TFE TATA PICTATAADBFDNARNA polEHTF IIIIH螺旋酶使在螺旋酶使在AAUAAA后继续转录出的后继续转录出的RNA脱离模板链，转录终止，脱离模板链，转录终止，RNA pol脱落，该脱落，该RNA被被RNase水解。水解。AAUAAAGUGUGUG5 AAUAAAployA

9、真核生物的转录终止真核生物的转录终止1323 processingGUGUGUG解螺旋酶解螺旋酶核酸酶核酸酶G1.3.剪接（splicing）（）（核内）成熟mRNA与基因DNA杂交电镜所见1043185 51129 118143156剪接方式剪接方式apoB mRNA的编辑加工的编辑加工 3 末端加尾（剪切内含子，拼接外显子）剪切内含子，拼接外显子）剪接hnRNAmRNA修饰修饰编辑编辑小结：小结：5 GATCTGACTGACATAGACATAGAT 3 coding(=non-template)strand3 CTAGACTGACTGTATCTGTATCTA 5 template stra

10、nd 5 GAUCUGACUGACAUAGACAUAGAU 3 mRNA 操纵子操纵子调控区调控区信息区信息区启动子启动子操纵基因操纵基因与与DNA结合后结合后促进转录促进转录，这种基因表达，这种基因表达调控的方式称为调控的方式称为正调控正调控。CAP蛋白蛋白(catabolic gene activator protein，CAP)分解代谢物基因活化蛋白分解代谢物基因活化蛋白ntrC蛋白蛋白 是大肠杆菌氮代谢基因激活蛋白，是大肠杆菌氮代谢基因激活蛋白，其自身活性可通过其自身活性可通过ntrB使其磷酸化（有活性）使其磷酸化（有活性）和去磷酸化（无活性）而被调节和去磷酸化（无活性）而被调节con

11、sensusTATA(Pribnow)boxE.coli Promoters Positive vs.Negative Regulation Allosteric EffectorsBinding can also be required for binding of repressor(e.g.Trp)or can block an activator.Organization of Lac Operon and LacIOperonpromoterRibosome initiationRegulation of Gene ExpressionIPTG also induces Splits

12、 lactose lactosetransport?（异丙基硫代半乳糖苷）半乳糖cAMPAMPACAdenylate cyclase and CAP mediate glucose repression of LacAdenylate cyclase(AC)is an enzyme that synthesizes cyclic AMP(cAMP)from ATPHigh glucose adenylate cyclase is inhibited(indirectly,via a catabolic product)Therefore cAMP levels are LOWAbsence o

13、f glucose adenylate cyclase is NOT repressedTherefore cAMP levels are HIGHcAMP forms a complex with the CAP protein,which allows it to then bind to the CAP site upstream of the Lac operon.Binding of the CAP protein is required to allow RNA polymerase to bind to the lac promoter and turn on transcrip

14、tion.In the absence of CAP binding,there is no(or very little)transcription of the lactose operon,even in the presence of lactose.glucoseCAP Binding Bends DNAvThis DNA bending results in more efficient RNA polymerase bindingCAP mediates glucose repression of LacPromotes transcriptionLactose Glucose-

15、+-+-+LacICAP-cAMPFour States of the Lac OperonCAPThe Arabinose Operon This loop prevents RNA transcription(NOT true for all loops)No Arabinose present,operon OFFArabinose present,Glucose absent,operon ONGenomic Organization of the Trp Operon Control of Gene Expression in the Trp Operon1)The enzyme c

16、atalyzing the first step in the pathway is inhibited by Trp(feedback control).2)In the presence of Trp,a repressor protein binds to an operator upstream of the Trp operon and shuts off transcription3)Attenuation.There is a 160 base pair region in the Trp mRNA that causes transcription to terminate p

17、rematurely if Trp is present.Trranscriptional ControlAttenuation ControlDNA Structure Major and Minor GroovesZinc Finger MotifHelix-Turn-Helix MotifLeucine ZipperLeucine ZipperZipper:every 7th residue is a Leu Hydrophobic interfaceH-Bonding in a Protein-DNA Interaction1.Northern印迹+2.狭缝印迹法3.组织原位杂交4.RNase保护测定法六、转录组学(transcriptomics)Global mRNA profiling4.Massively parallel signature sequencing (MPSS)