分子生物学CHGene Transcription andPPT学习教案

1、会计学1分子生物学分子生物学CHGene Transcription and GENETIC INFORMATION 1. REPLICATION(DNA SYNTHESIS) 2. TRANSCRIPTION(RNA SYNTHESIS) 3. TRANSLATION(PROTEIN SYNTHESIS)DNARNAPROTEIN第1页/共142页RNA polynucleotide chain 2 -OH makes 3, 5 phosphodiester bond unstableDNA polynucleotide chain第2页/共142页第3页/共142页有意义链有意义链无意义链

2、无意义链只针对已确定的某一基因而言只针对已确定的某一基因而言第4页/共142页第5页/共142页第6页/共142页第7页/共142页E.coli聚合酶亚基聚合酶亚基第8页/共142页Prokaryotic RNA polymerase structureRNA polymerase of E. coli is a multisubunit proteinSubunitNumberRole a a 2uncertain b b 1forms phosphodiester bonds b b 1binds DNA template s s 1recognizes promoter and faci

3、litates initiationa a2 2bbbbs sa a2 2bbbb + s sholoenzyme core polymerase sigma factor第9页/共142页第10页/共142页的延伸。的延伸。b b亚基亚基碱性极强，与碱性极强，与DNA模板模板结合的亚基；结合的亚基；第11页/共142页RNA转录的抑制剂转录的抑制剂第12页/共142页中有多种不同中有多种不同s s亚基；亚基；s s亚基在决定亚基在决定RNA聚合酶起始转录中起关键作用聚合酶起始转录中起关键作用第13页/共142页第14页/共142页由含由含s s70RNA多聚酶全酶识别的典型大肠杆菌启动子多聚

4、酶全酶识别的典型大肠杆菌启动子第15页/共142页第16页/共142页列列第17页/共142页启动子结构第18页/共142页Promoter structure in prokaryotes5PuPuPuPuPuPuPuPuAUGPromoter+1+20-7-12-31-365mRNAmRNATTGACAAACTGT-30 regionTATAATATATTA-10 region84 7953 45%82T TG64AC A79T44T96%T95A59A51Aconsensus sequences-30-10transcription start sitePribnow box+1第19页

5、/共142页-35区：共同序列为区：共同序列为TTGACA，是，是RNA聚合酶聚合酶起始识别区，这一识别过程起始识别区，这一识别过程与与s s因子有因子有关。关。第20页/共142页第21页/共142页上节回顾：上节回顾： 1. DNA损伤修复损伤修复 2. 基因转录基本概念基因转录基本概念, 特点特点 3. 原核生物原核生物RNA聚合酶聚合酶第22页/共142页RNA聚合酶全酶识别聚合酶全酶识别-35区，再识别区，再识别-10区，区，形成形成封闭型起始复合物封闭型起始复合物转录起始位点处转录起始位点处DNA解链，形成解链，形成开放型起始复合物开放型起始复合物第一个核苷酸结合上去（多数是第一个

6、核苷酸结合上去（多数是G/A、C）合成合成7-8个核苷酸，起始过程完成个核苷酸，起始过程完成第23页/共142页RNA polymerase holoenzyme (+ s s factor) closed promoter complex (moderately stable) the sigma subunit binds to the -10 region once initiation takes place, RNA polymerase does not need very high affinity for the promoter sigma factor dissociate

7、s from the core polymerase after a few elongation reactions elongation takes place with the core RNA polymerase open promoter complex (highly stable) the holoenzyme has very high affinity for promoter regions because of sigma factors s sigma can re-bind other core enzymesThe sigma cycles ss s第24页/共1

8、42页TranscriptionRNA polymeraseclosed promoter complexopen promoter complexinitiationelongationterminationRNA product第25页/共142页第26页/共142页RNA转录延伸期转录泡模转录延伸期转录泡模型型第27页/共142页第28页/共142页第29页/共142页终止子结构第30页/共142页新合成的新合成的RNA脱落。脱落。第31页/共142页不依赖Rho因子的转录终止第32页/共142页第33页/共142页依赖Rho因子的转录终止第34页/共142页终止子：提供转录终止信号的一

9、段终止子：提供转录终止信号的一段 DNA 序列。序列。 终止因子：协助终止因子：协助 RNA 聚合酶识别聚合酶识别 终止子的蛋白质辅助因子。终止子的蛋白质辅助因子。 第35页/共142页第36页/共142页子、组氨酸子、组氨酸操纵子操纵子组氨酸抑制了组组氨酸抑制了组氨酸操纵子氨酸操纵子的表达，避免把原料用于的表达，避免把原料用于不必要的合不必要的合成。）成。）第37页/共142页第38页/共142页lac IPOpromoter - operatorlac repressorlac Zlac Ylac AThe lactose operon in E. colib b-galactosidas

10、epermeaseacetylaseLACTOSE GLUCOSE + GALACTOSEb b-galactosidasethe function of the lactose (lac) operon is to produce the enzymes required to metabolize lactose for energy when it is required by the cell promoter binds CAP and RNA polymerase operator binds the lac repressor第39页/共142页第40页/共142页第41页/共1

11、42页解除葡萄糖的阻遏作用。解除葡萄糖的阻遏作用。第42页/共142页第43页/共142页第44页/共142页Regulation of the lactose operon - negative controllac IPOpromoter - operatorlac repressorlac IPlac Zlac Ylac A the repressor tetramer binds to the operator and prevents RNA polymerase from binding to the promoterlac Zlac Ylac ANO TRANSCRIPTIONR

12、NA pol RNA polymerase is blocked from the promoter第45页/共142页NO TRANSCRIPTION when lactose becomes available, it is taken up by the cell allolactose (an intermediate in the hydrolysis of lactose) is produced one molecule of allolactose binds to each of the repressor subunits binding of allolactose re

13、sults in a conformational change in the repressor the conformational change results in decreased affinity of the repressor for the operator and dissociation of the repressor from the DNAAlleviation of negative control - action of the inducer of the lac operonallolactoselac IPlac Zlac Ylac Alac IPlac

14、 Zlac Ylac A IPTG (isopropyl thiogalactoside) is also used as a (non-physiological) inducer第46页/共142页lac IPlac Zlac Ylac ANO TRANSCRIPTIONRNA polO repressor (with bound allolactose) dissociates from the operator negative control (repression) is alleviated, however. RNA polymerase cannot form a stabl

15、e complex with the promoter第47页/共142页lac IPOlac Zlac Ylac ARegulation of the lactose operon - positive control in the presence of both lactose and glucose it is not necessary for the cell to metabolize lactose for energy in the absence of glucose and in the presence of lactose it becomes advantageou

16、s to make use of the available lactose for energy in the absence of glucose cells synthesize cyclic AMP (cAMP) cAMP1 serves as a positive regulator of catabolite operons (lac operon) cAMP binds the dimeric cAMP binding protein (CAP)2 binding of cAMP increases the affinity of CAP for the promoter bin

17、ding of CAP to the promoter facilitates the binding of RNA polymerase 1 cAMP = 3, 5 cyclic adenosine monophosphateactive CAPinactive CAPcAMP+NO TRANSCRIPTION 2 also termed catabolite activator protein第48页/共142页lac Ilac repressorlac Zlac Ylac Ab b-galactosidasepermeaseacetylaseRNA polTRANSCRIPTION AN

18、D TRANSLATION OCCURinactive repressorActivation of lac operon transcription the function of the lactose (lac) operon is to produce the enzymes required to metabolize lactose for energy when it is required by the cell第49页/共142页第50页/共142页第51页/共142页第52页/共142页终止序列终止序列 因子因子第53页/共142页第54页/共142页酶酶酶活产 物RNA聚

19、合酶I 50-70%rRANRNA聚合酶II20-40%hnRNA（mRNA的前体）RNA聚合酶III 10%tRNA和snRNA第55页/共142页真核生物三种RNA聚合酶的比较第56页/共142页第57页/共142页RNA聚合酶聚合酶III：含锌蛋白质，由种亚含锌蛋白质，由种亚基组成。基组成。第58页/共142页RNA聚合酶中最高的。聚合酶中最高的。rDNA40sRNA18s、5.8、28srRNA加工加工RNA pol I转录转录第59页/共142页第60页/共142页rRNA前体前体第61页/共142页第62页/共142页5端转录终止序列端转录终止序列包含了起始位点，决定转录起始包含了

20、起始位点，决定转录起始明显增强核心启动子的转录活性，明显增强核心启动子的转录活性，决定转录强弱决定转录强弱第63页/共142页第64页/共142页非洲爪蟾非转录区的结构非洲爪蟾非转录区的结构第65页/共142页录起始频率），启动子的强弱起决于录起始频率），启动子的强弱起决于UPE的类型。的类型。决定转录起始的选择，绝大多数真核基因决定转录起始的选择，绝大多数真核基因正确表达所必需正确表达所必需确定真正的起始位点确定真正的起始位点第66页/共142页第67页/共142页Sequence elements within a typical eukaryotic gene1GCTATACAATGC-

21、25-50-80-95-1301 based on the thymidine kinase gene octamertranscription elementpromoterTATA box (TATAAAA) located approximately 25-30 bp upstream of the +1 start site determines the exact start site (not in all promoters) binds the TATA binding protein (TBP) which is a subunit of TFIIDGC box (CCGCC

22、C) binds Sp1 (Specificity factor 1)CAAT box (GGCCAATCT) binds CTF (CAAT box transcription factor)Octamer (ATTTGCAT) binds OTF (Octamer transcription factor)+1ATTTGCAT第68页/共142页指导指导RNA合成；合成；增强子：若干元件作为整体促进远增强子：若干元件作为整体促进远端的转录。端的转录。第69页/共142页RNA聚合酶II启动子第70页/共142页3.大多为重复序列，一般大多为重复序列，一般50bp；第71页/共142页一个增

23、强子并不限于促进某一一个增强子并不限于促进某一特殊启动子的转录，它能刺激附近特殊启动子的转录，它能刺激附近的任一启动子。的任一启动子。第72页/共142页第73页/共142页第74页/共142页基因内启动子基因内启动子基因外启动子基因外启动子混合启动子混合启动子第75页/共142页第76页/共142页第77页/共142页5S rRNA基因启动子在转录区内基因启动子在转录区内第78页/共142页元件。元件。如：海胆硒代半胱氨酸如：海胆硒代半胱氨酸tRNA基基因因第79页/共142页第80页/共142页转录转录因子因子通用转录因子普遍性转录因子通用转录因子普遍性转录因子主要与启动子核心元件框结合，

24、主要与启动子核心元件框结合，是同类基因转录都必须的转录因子，是同类基因转录都必须的转录因子， 如如TFA、B、D、E等。等。转录调控因子转录调控因子与上游调控区和远端调控区（增强子）结合，与上游调控区和远端调控区（增强子）结合，影响转录水平，在一些特定的基因转录中起作用。影响转录水平，在一些特定的基因转录中起作用。（不同基因的特殊转录调控因子）（不同基因的特殊转录调控因子）转录因子：以反式作用影响转录的因子。转录因子：以反式作用影响转录的因子。第81页/共142页第82页/共142页亮氨酸拉链模型及它在转录因子两个分子二聚体化中的作用亮氨酸拉链模型及它在转录因子两个分子二聚体化中的作用第83页

25、/共142页二聚体化转录因子二聚体化转录因子C/EBP的亮氨酸拉链的亮氨酸拉链和和DNA结合结构域的结构模型结合结构域的结构模型第84页/共142页第85页/共142页第86页/共142页UBF与与UCE结合，结合，SL1与与UBF结合，使结合，使UBF向前移动向前移动改善改善UBF与核心元件结合，形成一个跨越核心与核心元件结合，形成一个跨越核心元件元件UCE的蛋白质的蛋白质-DNA复合体复合体RNA聚合酶聚合酶I结合上去，形成开放性起始复合物结合上去，形成开放性起始复合物转录开始转录开始第87页/共142页A框框TF BTF CTF ARNApol 激活因子激活因子增强子结合因子增强子结合因

26、子使使RNApol 正确正确定位于起始位点定位于起始位点第88页/共142页第89页/共142页 类型类型基因转录因子基因转录因子 TF A、B、D、E等等第90页/共142页类型类型基因调控区域与各种转录因子的结合位置基因调控区域与各种转录因子的结合位置第91页/共142页第92页/共142页类型类型基因转录的起始基因转录的起始第93页/共142页第94页/共142页第95页/共142页第96页/共142页第97页/共142页剪接剪接RNA编辑编辑第98页/共142页功能的发挥；功能的发挥；第99页/共142页Steps in mRNA processing (hnRNA is the pr

27、ecursor of mRNA) capping (occurs co-transcriptionally) cleavage and polyadenylation (forms the 3 end) splicing (occurs in the nucleus prior to transport) exon 1 intron 1 exon 2capcapcappoly(A)cappoly(A)Transcription of pre-mRNA and capping at the 5 endCleavage of the 3 end and polyadenylationSplicin

28、g to remove intron sequencesTransport of mature mRNA to the cytoplasm第100页/共142页n对翻译起识别作用对翻译起识别作用（ m7G5ppp5Np 优先与优先与核糖体结合）。核糖体结合）。第101页/共142页三种帽结构三种帽结构第102页/共142页 三种帽结构三种帽结构第103页/共142页初级转录物的切除与多聚腺苷酸化第104页/共142页Capping occurs co-transcriptionally shortly after initiation guanylyltransferase (nuclear)

29、 transfers G residue to 5 end methyltransferases (nuclear and cytoplasmic) add methylgroups to 5 terminal G and at two 2 ribose positions onthe next two nucleotides capping involves formation of a 5- 5 triphosphate bond cap function protects 5 end of mRNA (increases mRNA stability) required for init

30、iation of protein synthesispppNpNmGpppNmpNm第105页/共142页Polyadenylation cleavage of the primary transcript occurs approximately 10-30 nucleotides 3-ward of the AAUAAA consensus site polyadenylation catalyzed by poly(A) polymerase approximately 200 adenylate residues are added poly(A) is associated wit

31、h poly(A) binding protein (PBP) function of poly(A) tail is to stabilize mRNAmGpppNmpNmAAUAAAmGpppNmpNmAAUAAAAAAAAA3cleavagepolyadenylation第106页/共142页切除内含子成为切除内含子成为mRNA，进入细胞，进入细胞浆内。浆内。第107页/共142页 剪接体剪接体SnRNP参与参与mRNA 酶蛋白参与的剪接酶蛋白参与的剪接tRNA第108页/共142页b-珠蛋白的mRNA与基因杂交 b-珠蛋白的pre-mRNA与基因的杂交第109页/共142页鸡卵清蛋白鸡

32、卵清蛋白mRNA与基因杂交图与基因杂交图第110页/共142页核酸酶核酸酶Ribozyme具有催化活性的具有催化活性的RNA分子。分子。第111页/共142页GroupI内含子剪接第112页/共142页四膜虫四膜虫rRNA的自我剪接的自我剪接第113页/共142页第114页/共142页Chemistry of mRNA splicing two cleavage-ligation reactions transesterification reactions - exchange of onephosphodiester bond for another - not catalyzed byt

33、raditional enzymes branch site adenosine forms 2, 5 phosphodiester bondwith guanosine at 5 end of intronG-p-G-U A-G-p-G2OH-A-53intron 1exon 1exon 2Pre-mRNAFirst clevage-ligation (transesterification) reactionbranch site adenosine第115页/共142页G-OH 3 A-G-p-GU-G-5-p-2-A53AAO-G-p-G53U-G-5-p-2-A A3 G-ASpli

34、cingintermediateLariatexon 1exon 1exon 2exon 2intron 1intron 1Second clevage-ligation reactionSpliced mRNA ligation of exons releases lariat RNA (intron)第116页/共142页GroupII内含子剪接第117页/共142页细胞核小分子核糖核蛋白细胞核小分子核糖核蛋白snRNP第118页/共142页真核生物内含子真核生物内含子5剪接位点和剪接位点和3剪接位点的保守顺序剪接位点的保守顺序第119页/共142页第120页/共142页剪接过程剪接过程：

35、U1 与与5剪接点结合，剪接点结合， U2与分支点结合，与分支点结合，U4/ U6 复合物结合，形成完整的复合物结合，形成完整的 “剪接体剪接体” 5剪接点被切开，形成剪接点被切开，形成5-外显子和套索中间产物外显子和套索中间产物U4 离开剪接体，离开剪接体， 3剪接点切开剪接点切开两个外显子共价连接，形成成熟两个外显子共价连接，形成成熟mRNA和套索状内含子和套索状内含子第121页/共142页Recognition of splice sites invariant GU and AG dinucleotides at intron ends donor (upstream) and acc

36、eptor (downstream) splice sitesare within conserved consensus sequencessmall nuclear RNA (snRNA) U1 recognizes thedonor splice site sequence (base-pairing interaction) U2 snRNA binds to the branch site (base-pairing interaction)Y= U or C for pyrimidine; N= any nucleotide G/GUAAGU.A.YYYYYNYAG/Gdonor

37、(5) splice siteacceptor (3) splice sitebranch siteU1U2第122页/共142页Spliceosome - assembly of the splicing apparatus snRNAs are associated with proteins (snRNPs or “snurps”) splicing snRNAs - U1, U2, U4, U5, U6 antibodies to snRNPs are seen in the autoimmunedisease systemic lupus erythematosus (SLE)G-p

38、-G-U A-G-p-G2OH-A-53intron 1exon 1exon 2Spliceosome assemblyStep 1: binding of U1and U2 snRNPsU1= hnRNP proteinsU2第123页/共142页G-p-G-U A-G-p-G2OH-A-53intron 1exon 1exon 2Step 2: binding of U4, U5, U6U1U5U2U4 U6G-p-G-U A-G-p-G2OH-A-53intron 1exon 1exon 2Step 3: U1 is released,then U4 is releasedU5U2U6第

39、124页/共142页G-p-G53U-G-5-p-2-A A3 G-Aintron 1mRNA2OH-AU5U2U6Step 4: U6 binds the 5 splice site andthe two splicing reactions occur,catalyzed by U2 and U6 snRNPs第125页/共142页SnRNA参与的参与的mRNA前体的剪接前体的剪接第126页/共142页自我剪接与剪接体催化的剪接比较自我剪接与剪接体催化的剪接比较第127页/共142页第128页/共142页tRNA的前体加工的前体加工第129页/共142页特异性特异性RNA内切酶识别内含子二

40、级结构，内切酶识别内含子二级结构，将两侧磷酸二酯键切开，形成将两侧磷酸二酯键切开，形成5-OH和和3-P；（不需；（不需ATP）两个两个tRNA半分子碱基互补配对，形成半分子碱基互补配对，形成成熟的成熟的tRNA构象，构象，RNA连接酶形成磷连接酶形成磷酸二酯键（需要酸二酯键（需要ATP）第130页/共142页第131页/共142页第132页/共142页鸡卵清蛋白前体鸡卵清蛋白前体mRNA到成熟到成熟mRNA的过程的过程第133页/共142页大鼠中降钙素基因转录子的加工大鼠中降钙素基因转录子的加工第134页/共142页剪接错误造成剪接错误造成b b贫血病贫血病第135页/共142页Mutati

41、ons that disrupt splicing bo-thalassemia - no b-chain synthesis b+-thalassemia - some b-chain synthesisNormal splice pattern:Exon 1Exon 2Exon 3Intron 1Intron 2Donor site: /GUAcceptor site: AG/Intron 2 acceptor site b bo o mutation: no use of mutant site; use of cryptic splice site in intron 2Exon 1Exon 2Intron 1mutant site: GG/Intron 2 cryptic acceptor site: UUUCUUUCAG/GTranslation of the retained portion of intron 2 results in prem