林木分子-RNA的转录.ppt

Click to edit Master title style,*,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,第,4,章,RNA,转录,(,RNA transcription,),4.1,转录的基本概念,4.2,转录起始,4.3,转录延伸,4.4,转录终止,4.5 RNA,的加工,本章内容,基因表达的第一步,模版链,（反编码链,/,反义链,/,无义链,/,负链）,在,依赖,DNA,的,RNA,聚合酶,的作用下,模板单链,DNA,的极性方向为,3 5,而非模板单链,DNA,的极性方向与,RNA,链相同，均为,5 3,(,书写,DNA,序列时，仅写非模板序列，可不注明极性方向,),按,A U,，,C G,配对的原则，合成,RNA,分子,以,D.S.DNA,中的一条单链作为转录的模板,极性：,转录的效率与转录单位的位置有关，这个特点称为,转录的极性。,4.1,转录的基本概念,DNA,3,-,TACTCAT,-5,RNA 5,-,AUGAGUA,-3,5,-,ATGAGTA,-3,Non-template(sense strand),template(antisense strand),RNA,的转录包括,promotion,elongation,termination,三过程,转录单位,（,transcriptional unit,）,：,从启动子（,promoter,）到,终止子（,terminator,）。,原核,生物中的转录单位多为,polycistron,in,operon,真核,生物中的转录单位多为,monocistron,no,operon,转录原点记为,+1,，其上游记为,负值,，下游记为,正值,+1,-10,+10,upstream,start point,TSS,downstream,4.2,转,录,起,始,Promoter,的结构与功能,(,Prok,.,E.coli,),a)promoter,由两个重要部分组成,-170 -60,Fis,site;,binding sites for the transcription-activator protein,Fis,.,Fis,site,do not bind to,RNA polymerase,are,not classical promoter,elements,but instead are members of enhancers.,-60-70 -40 up element,CAP,cAMP,binding site,-35 -10 core promoter,RNApol,.binding site,基因表达调控的正控制位点,Promoter region identified by,DNAaseI,footprinting,method,RNA polymerase (no NTP),DNA sequencing,DNase,I,digestion DNA,+,dissolve,原核生物启动子发现的,DNA,足迹实验,Core promoter region including,Sextama,Box,RNApol,.recognition site(,R,site),TTGAC(,Sextama,Box),-35 site,RNApol,.,loosely binding site,Pribnow,Box,TATAAT(,pribnow,Box),-10 site,RNApol,.,firmly binding site,(,B,site),Initiation site,+1 RNA transcriptional,startpoint,(,I,site),A/G,-35,(R),-10,(B),+1,(I),RNA,R,-35,B,-10,I,+1,Sextama,Box,Pribnow,Box,Initiation,Core promoter region including,R,site:recognition,B,site:binding,I,site:initiation,Sextama,Box,与,Pribnow,Box,间距,17bp,有利于,RNApol,启动,Core Enzyme,Holo,Enzyme,for elongation,for initiation,依靠静电作用力,依靠空间结构,非专一性与非特异,DNA,结合,专一性与,特异,DNA,结合,cover60bp,原核生物,RNA,聚合酶的亚基成分,因子,促使,RNApol,与,DNA,模板链,上游因子,结合,位于前端的,因子使双链解链为单链,位于尾端的,因子使单链新聚合为双链,核心酶的组建因子,+,2,+,+,因子,促进,RNA,pol,+NTP RNA elongation,完成,NMP,之间的磷酸脂键的连接,Editing,因子,促使,RNA polymerase,与,非模板链（,sense strand,）结合,因子,(sigma factor),重复使用,(Re-usable),使,Holo,-enzyme,识别,Sextama,Box,与模板链结合,修饰,RNApol,构型,降低全酶与,DNA,的,非专一性,结合力,增强全酶与,R,B site,的专一性结合力,导致,RNA,链的延伸缓慢,原核生物,RNApol,(Core),的结构与功能,Enzyme Movement,DNA coding strand(,),Rewinding point(,),Unwinding point(,),RNA binding site,RNA/DNA hybrid(,),DNA template strand,Core Enzyme,使,DNA,形成,10-17bp,的解链区,亚基离开全酶后可以重复使用,l,基本水平转录的,cis,-factor,(promoter or basic factor),Core promoter,(Cap site,TATA box,必需因子,),UPE,(Upstream Promoter Element)(CAAT box,GC box),For housekeeping gene,(constitutive expression),l,特异诱导高效表达的,cis,-factor,Enhancer,For luxury gene,(inducible expression),与转录启动相关的,cis,-factors,组成型表达,诱导型表达,持家基因,Promoter for basic transcription,(class II recognized by RNA polymerase II),l,Cap site;initiation point(+1),Capping,m,7,Gppp,A/G-,70,-AUG-(in mRNA),1-4Kb -70 -30 +1,GC island CAAC/,T,box TATA box,Cap,Enhancer UPE core promoter,Promoter(basic factor),Initiation region,PyPy,A,NT/(A)PyPy,l,TATA box/,Hogness,box/Goldberg-,Hogness,box(-30),Rich AT and rich GC flanked,rich GC-,-T A T A A (,T,)A A (,T,)-,rich GC,82 97 93 85 63,(,37,),83 50,(,37,),1-4Kb -70 -30 +1,GC island CAAC/,T,box,TATA box,Cap,Enhancer UPE core promoter,Promoter(basic factor),l,GC island(C no,methylated,)and CAAT box(,CAT box,UPE)(-70),GCGC,-GGC(,T,),CAAT,CT-,Response to effect of transcription,Range 30,bp,1-4Kb -70 -30 +1,GC island CAAC/,T,box,TATA box Cap,Enhancer UPE core promoter,Promoter(basic factor),l,Promoter functions,CAAT box,(modulator),controlling transcriptional efficiency,(GC island!),TATA box,(selector),controlling starting point and efficiency,30bp from TATA box to I site is required,I site,(initiator),determination initiation point and capping of mRNA,Cis,-factor for inducible expression,Special box,Arabidopsis,I box(GATAAG),-5-20-G box-,Pea,H box(CCTACC),-5-20-G box-,modul,HSE,(,Heat Shock,Element),GRE,(,Glucocoricoid,Response Element),糖皮质激素应激元件,MRE,(,Metal,Response Element),TSE,(,Tissue,Special Element),l,Inducible expressed by environment,Ig,ATG,G,AAAT+Oct-2 factor B,细胞表达,GH,ATG,A,A,T,AT +Pit-1 factor ,脑下垂体表达,Hotspot,Enhancer,Enhance expression Basic expression,Position not be fixed Isolated region,Bi-directional element Mono-directional element,Enhancer Promoter,No for special gene only for special gene,Enhancer,与,Promoter,的比较,Chambon,discovered the first enhancer in the 5-flanking region of the SV40 early gene.,Enhancer,的结构与功能,-Enhancer,由两个以上的,增强子成分,(,Enhancer Element,),组成,-Enhancer Element,必需由两个紧密相连,具有间距效应的,增强子元,(,Enhanson,）,组成,-,各个,Enhanson,(,cis,-factor),与激活蛋白,(,trans-factor),结合,增强特异性转录,促进转录的复合体,+,UPE+core promoter,基本转录复合体,Enhancer,Specifical,transc,.Factor(,tran,-factor),Specifical,transc,.Factor(,tran,-factor),+,+,Activation domain of,Enhancer complex,Activation domain of,Enhancer complex,Enhancer Element,Enhancer Element,(100bp),Enhanson,(,cis,-factor),Enhanson,(,cis,-factor),(1 kb)to modulate transcription,Somehow cause the chromatin to coil up into a,Condensed,Inaccessible,Inactive form,Thereby,preventing transcription of neighboring genes,一种通过一段延伸的,DNA,区域影响染色质结构,从而调节转录关闭的,DNA,元件。,Insulator:,(,绝缘子,),Elements that prevent the passage of activating or inactivating effects,能够阻断激活或失活效应通过的元件,Insulator(,绝缘子,),具有两种功能,a)When an insulator is placed,between enhancer&promoter,it prevents the enhancer from activating the promoter,Insulator(,绝缘子,),具有两种功能,b)When an insulator is placed,between active gene&heterochromatin,it provides a barrier that protects the gene against the inactivating effect that spreads from the heterochromatin,与基本转录相关的,T.F.,General transcription factors,真,核生物,中,RNApol,的作用必须有其他相关转录蛋白在启动子处的先期结合才能启动转录,RNApol,.I,II,III,L,with 78%homologous,between 3,RNApol,.I,II,III,L,of,prokaryote,RNApol,.&,L,(,功能相似,),Including L,L subunit&,7-12,small subunits,进,化,渊,源,RNA polymerase in Eukaryotes,与基本转录相关的,RNA,pol,&T.F.,真核生物,原核生物,RNApol,II,-For pre-mRNA transcription,-Located in,nucleoplasm,-L,maximum subunit 240,kD,&have specific COOH-end,named,CTD,(Carboxyl Terminal Domain)only in,RNApol,II,CTD-end,：,7aa repeats&high frequency,phosphorylation,Tyr,Ser,P,Pro,Thr,P,Ser,P,Pro,Ser,P,与基本转录相关的,T.F.,（转录因子）,General transcription factors,(GTF),GTF for basic transcription,l,TF(I,II,III),T,ranscriptional,F,actor,l,TBP(,T,ATA box,B,inding,P,rotein),l,TAF(,T,BP,A,ssociate,F,actor),l,RAP(,R,NApol,.,A,ssociate,P,rotein),Cis,-factor,+,TIC(Transcriptional Initiate Complex),-,RNApol,II +,20TFIIs,逐级组装,TIC,转录启动,(Transcriptional Initiation Complex),TATA +1,Wilds minor groove,TFII A,TBP of D,pre-TIC,TFIIA,：,Binds to TFII D,Enhances TFII D binding to TATA box,Stabilizing the DNA-TFII D complex,(TBP),：,needed for,RNApol,I,II,III,TFIID,：,A sort of protein complex(TBP&8 TAF),Very high conserved,C-end domain of 180,aa,Binds with DNA in,minor groove&wilds it,Determination,initiation starting site,Control UPE effect,for basic transcription,TFIIB:,Once TFIID has bound to DNA,TFIIB binds to TBP of TFIID and RNA pol.II,TFIIB as a bridging factor,allowing recruitment,of,RNApol,II to TIC together with RAP30 of TFIIF,TFIIF:,Including two subunits of,RAP30,RAP74,Binding and,recruiting,RNApol,II to assemble TIC,Promoting RNA elongation by its,helicase,(,ATPase,),TFII F,RNApol,II,TFII B,Basic TIC,bridging factor,helicase,(,ATPase,),TFIIH,:Large complex made up of 5 subunits,Helicase,activity(,ATPase,RAD25,),Kinase,activity,：,Phosphorylation,of CTD,DNA repair,TFIIE:,Associate with TFIIH kinase,Complete TIC,E,H,Phosphorylation,of CTD of,RNApol,II,H:,解旋酶和蛋白激酶活性,transcription activator,(activator,STF,),for gene-specific inducing transcription,character,of,activator,l,多为变构蛋白，具有,2,或,4,个独立的功能结构域（,domain,）,DNA-binding domain,Transcription regulation domain,activation/repression,domain,DNA-binding domain,Dimerization,domain (in some,dimer,factor),Transcription activation domain,Independence,of DNA-binding domain,and,transcription-activation domain,Transcription factor,reporter,Xp,interaction with,Yp,Screening,Yeast two-hybrid assay,将研究的,cDNA,与,AD,，,BD,分别构建两个表达载体转化酵母测验体系（商业化产品）,（,Y2H,）,activator,的活性调节,受到严格的,信号因子,的调控,signal,一般状态,（无活性）,binding DNA,变构,活化状态,激活转录,种类与结构特点,Cys/Cys,Cys,/His,Zinc finger,DNA-binding domain,Helix()-turn-Helix(,),(HTH),HelixloopHelix,(HLH or,bHLH,),dimerzation,domain,Leu,zipper,(,bZIP,),转录因子的结构特点,Acidic domain,Gln,-rich domain,Pro-rich domain,Activation domain,30,aa,/copy one finger,Phe(F,),Leu(L,),Tyr(Y,),core of hydrophobic,aa,9 direct repeats 30aa/copy,2Cys/2His,or,2Cys/2Cys,types,2Cys(2C)Zn 2His(2C),to form finger of 1,-helix&1-,sheet,Peptide chain,Zinc finger,C,C,H,H,转录因子以三种方式发挥,促进转录,的功能：,（,1,）,改变,DNA,构象,，使原来包埋在螺旋内部的启动子序列暴露出来，有利于,RNA,聚合酶与启动子的结合。,（,2,）,影响,RNA,聚合酶，,TF,因子与,DNA,结合形成,Basic transcription complex,，加强,/,减弱其特异性结合。,（,3,）参与其它调节因子之间的,interaction&activity,4.3,转录延伸,始于第一个磷酸二酯键的形成,伴随着,DNA,分子和酶分子构象的变化,In,Prok,.,起始时，,因子有利于,和,亚基具有与,DNA,专,一,性结合所要求的构象,起始后,因子解离,和,亚基构象发生变化,In,Euk,.,多种辅助因子的共同作用来保证这一转变,起始到延伸的转变,磷酸二酯键：指一分子磷酸与两个醇（羟基）酯化形成的两个酯键。,酶与产物,RNA,不解离,底物,NTP,不断加到,RNA,链的,3,-OH,端,形成一个磷酸二酯键后，核心酶向前滑动,延伸位点不断地接受新的,NTP,，,RNA,链不断延伸,延伸过程,（,Prok,）,始终保持三元复合物的结构,a),转录泡,RNApol,结合和转录的,DNA,模板区域，有,17bp,左右,DNA,形成解链区,转录泡处杂交双链很短,b),拓扑学问题,RNA,合成过程中，转录泡两端要发生拓扑转变,RNApol,的前沿,.,解螺旋作用,RNApol,的,后端,.DNA,的螺旋化,（保持解链区长度约,17,核苷酸左右）,超缠问题的解决靠DNA旋转酶,RNA-DNA杂交链也要求作旋转运动,c),转录过程中的延宕,1,）,RNA,的合成速度大约,30,50,个核苷酸秒,与蛋白,质的合成速度相近,(15,个,AA,sec),2,）模板,DNA,中,G,C,的存在,（,G,C,后的,810,个核苷酸）,延宕作用在,RNA,链的终止和释放中起重要作用,4.4,转录终止,Transcription termination,概念,Terminator,种类,基因末端终止子,(,terminator,),基因内部终止子（,attenuator,衰减器,）,Rho-independent,terminator,/Rho-dependent,terminator,Difficult to identify the primary transcript,l,RNApol,leave from terminator,In prokaryotes,In Eukaryotes,l,RNA released from Triplex(RNA,DNA,RNApol,),l,different from discontinue/read-through,because pre-mRNA processing,Rho-independent terminator,(simple terminator),Palindromic,sequence,G/C rich region that form hairpins of varying lengths in RNA,G/C rich hairpin are followed by a run of U residues in RNA,a),Structure,Rho-independent terminator,(simple terminator),回文结构,地满红花红满地,inhibited by K factor,RNApol,&DNA separated,b)Function,G/C rich,transcription delay,NusA,-protein,RNApol,pausing,RNA stem-loop&,polyA,/U,RNApol,leave,a),Structure,l,Palindromic,sequence,l,G/C rare in,palindrom,but,form loose hairpin in RNA,l,palindromic,sequence are not,followed by poly A/T in DNA,Rho factor be needed for,termination,Rho-dependent terminator,b)Function,RNApol,+,NusA,-protein,RNApol,pausing 60 more or less,or,read-through,termination,K,Rho,c)Rho-factor(),l,Hexamer,55kd neutral,helicase,-like,l,Contest RNA 3-end with,factor,l,ATPase,-like,GTPase,-like,activity need spacer of,50,Nt,l,转录终止效率取决于紧随回文的下游序列,-,保证,RNA/DNA,具有一定松弛的结合力,-,过低的结合力 成为,Rho-independent terminator,-,过高的结合力 引起,RNApol,read-through,-,依赖,Rho,因子的终止子间的效率差异取决于,终止子区,DNA/RNA,间结合力的差异,注：,factor,促进,RNA,pol,+NTP RNA elongation,model of rho-dependent termination,(b)When the hairpin forms in,the transcript,the polymerase,pauses,giving Rho a chance to,catch up.,(c)Rho,helicase,unwinds the,RNA-DNA hybrid and,releases the transcript.,.,Rho binds to the transcript,and pursues the polymerase.,4.5 RNA,的加工,1)RNA,的加工,将各种前体,RNA,分子加工成成熟的、有活性的,RNA,的过程,2),场所,主要是在细胞核内进行,3)RNA,加工的类型,a),剪切,：就是剪去部分序列；,b),剪接,：是指剪切后又将某些片段连接起来。,c),末端添加核苷酸,：如,tRNA,的,3-,末端添加,-CCA,。,d),修饰,：在碱基及核糖分子进行化学修饰。,e),RNA,编辑,：某些,RNA,，特别是,mRNA,自,DNA,模板上所获得的遗传信息，在转录作用后又发生了变化,。,tRNA,的加工,原核,tRNA,的加工,a),“,斩头,”,:,形成,5,末端；,（,RNaseP,）,b),“,去尾,”,:,切除,3,多余的核苷酸直至,CCA,，,形成,3-OH,末端,（,RNaseD,）,；,c,),修饰,：甲基化等,真核,tRNA,的加工和原核的区别,a),真核,tRNA,前体中无二聚体和多聚体；,b),增加了剪接内含子的过程；,c),真核生物,CCA,多由,tRNA,核苷酸转移酶,添加，而原核多由,tRNA,基因编码。,真核生物前,mRNA,加工过程,5,端加帽,（,5-capping,）,3,端加尾,剪接,（,splicing,）,甲基化修饰,（,methylation,）,前,mRNA,加工过程,三种,5,帽结构形式,帽,0,m7,GpppNpN,帽,1,m7,GpppN,m,pN,帽,2,m7,GpppN,m,pN,m,5,端加帽,（,5-capping,）,1,）防止被,5,外切核酸酶降解,2,）翻译时可被起始因子和核糖体识别,3,）有助于,mRNA,越过核膜，进入胞质,5,帽的作用,m,7,G(5-5,mRNA,guanyltransferase,鸟苷转移酶,),Base 1:2-OH,甲基化；,A(N6-,甲基化,),Base 2:2-OH,甲基化,5-capping,3,端加尾,poly(A,),聚合酶,-,Poly(A,),polymerase(PAP,);,poly(A,),：大部分真核,mRNA,有,poly(A,),尾巴,poly(A,),：无,poly(A,),尾巴，如组蛋白的,mRNA,加尾的作用,1,）,稳定,mRNA,2,）有助于,mRNA,从核到细胞质转运,3,端,:,poly(A,),3,末端多聚,A,尾的生成机理,（,1,）,识别因子,识别,AAUAAA,（,2,）剪切因子在加尾位点,AAUAAA,下游11,30,nt,处,剪切,RNA,；,（,3,）,poly(A,),聚合酶,合成,poly(A,),尾巴；,AAUAAA,(20bp),CA,5,UUGUGUGUUG,3,Polyadenylation,signal,Cleavage site,polyA,addition site,GC-rich region,内含子的剪接,核酶,(,Ribozyme,),是指本质为,RNA,或以,RNA,为主含有蛋白质辅基的一类具有催化功能的物质。,发现,1981,年,T.Cech,和,S.Atman,四膜虫,rRNA,类型,剪切型,：,相当于,内切核酸酶,剪接型,：,相当于,内切核酸酶及连接酶,其他类型,：如核苷酸转移，脱磷酸作用,前,mRNA,的内含子,:,GU-AG,类（主要）,G,100,U,100,A,62,A,68,G,84,T,63,12Py N,C,65,A,100,G,100,A,64,G,73,N,内含子,外显子,外显子,左位点,(5/,供体,),右位点,(3/,受体,),分支部位的共同序列,Py,80,N Py,80,Py,87,Pu,75,A,Py,95,前,mRNA,的,剪接（,splicing,）,GT-AG rule(,GT-AG,规则,),：指在,核,基因,内含子,开,始,和结,束,出现的,两,个,固,定的,脱,氧,核苷酸,:,5,端为,GT,3,端为,AG,，与前体,RNA,内含子剪接有关,前,mRNA,的内含子,:,AU-AC,类（次要）,1.,内含子自我催化断裂和连接（,ribozymes,）,2.,需要带有,3-OH,的鸟苷参与,3.,转酯反应,(,transesterification,),I,型内含子剪接的特点,I,型内含子的剪接,结构特点,无,GT-AG,结构,边界序列为,5 U,.3G;,II,型内含子,介于,GT-AG,和,I,型内含子之间的类型,结构特点,1,）边界序列为,5,GU,GCG,Yn,AG,；,2,）分支点顺序（,branch-point,seguence,）,保守序列,Py,Pu,Py,Py,T,A,Py,3,）分子内配对：,分支点顺序的中间和两侧各有,3-5bp,的序列与上游序列互补（,A,除外）,RNA,的编辑,RNA,的编辑,（,RNA editing,）,的概念,是指转录后的,RNA,在编码区发生碱基的加入，,丢失或转换等现象,b),编辑的生物学意义,1,、校正作用,2,、扩充遗传信息,机制：脱氨基作用的结果（胞苷和腺苷脱氨酶催化）,进行编辑的,脱氨酶,能特异性识别靶位点,c),编辑的机制,1),碱基转换,CAA,U,AA,肝脏,肠,哺乳类载脂蛋白,apo,-B,转录物的编辑,2),碱基插入,GGGGAAAGAUAUUGAUGAAAGGA,G,U,GG,U,GAA,UUU,A,UU,G,UUU,AUAUUGA,U,UG,U,A,UU,A,U,AGG,UU,A,AAA,UUU,A,U,G,UU,G,U,C,U,UUUAAC,UUUAAAUAUAAUAGAAAAUUG,AAA,A G G C,UUUAAC,UUUAAAUAUAAUAGAAAAUUG,编辑前,RNA,指导,RNA,指导,RNA,的作用模式,利什曼原虫细胞色素,b mRNA,的编辑,编辑后,RNA,指导,RNA,编辑的反应：,切割,插入,连接,本章要点,1.,名词解释：,增强子，,RNA,编辑，终止子的回文结构,2.,简述：,真核生物转录的起始方式,转录因子发挥功能的方式,RNA,加工的类型,pre-mRNA 5,端加帽和,3,端加尾的机理与功能,GU-AG,类,pre-mRNA,内含子剪接方式,Rho,依赖型转录终止方式,