分子生物学-第四章-Transcription--Elongation.ppt

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,*,After 100 Nt,l,模板DNA中G/C的存在 delay,l,promoter clearance elongation complex,TFII E,H,ATP,Negative-superhelix needed,l,RNApol II+TFII F（,helicase,）Transcription,55kd TFII J stimulation elongation,4.3.1 Transcription delay,(转录过程的延宕,dang,）,4.3,Transcription Elongation,4.3.2 Pre-mature termination of transcription,RNA pausing,形成系列,调节机制,以降低非生理性，非特异性的基因表达,由于核小体致密的结构,l,3-end 具有高亲和力结合的核蛋白质影响,l,Silencer or DNA loop 结构的影响,l,DNA分子的弯曲,延伸过程与两种transcription Complex 性质相关,l,进行型复合体（processive complex）,RNApol II stop at 3-end of gene,并表现为DRB,S,(5,6-2氯-1-D呋喃核糖苯并咪唑),(5,6-dichloro-1-D ribofuranosyl benzimidazole),被激活的基因的表达方式，其RNA能顺利延伸,l,非进行型复合体（Non-processive complex）,未被激活的基因的表达方式,RNApol II pausing at 5-end of gene，并表现为,DRBR,-是对基因非激活转录的中止机制,-维持正常生理代谢进行的机制,-,与转录启动复合体的非完整性有关（abortive TIC）,Terminator:,A sequence of DNA,presented at the end of the transcript,that causes RNA polymerase to terminate transcription.,4.4 Transcription termination,4.4.1,Termination,i,n,prokaryotes,Palindromic,sequence,G/C rich region that forms hairpins of varying lengths(7-20bp)in RNA,G/C rich hairpin are followed by a run of U residues in RNA,Structure,Rho-independent,terminator,(Intrinsic terminator),Function,G/C rich transcription delay,RNA stem-loop&,polyA,/U,RNApol,leave,inhibited by K factor,RNApol,&DNA separated,因子：,强碱性亚基,促使,RNA pol,与非模板链（,sense,strand,）,强,结合,受K酶抑制,Structure,l,Palindromic sequence,l,no G/C rich 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,Rho-factor(),l,Hexamer 55kd neutral,l,Contest RNA 3-end with factor,l,ATPase-like,GTPase-like activity needs 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,-各依赖 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,The action of rho factor may create a link between transcription and translation when a rho-dependent terminator lies soon after a nonsense mutation.,Antitermination,depends on specific sites,4.4.2 Anti-termination in Rho-dependent,terminator of Prokaryotes,Antitermination proteins:,allow RNA polymerase to transcribe through certain terminator sites.,Switches in transcriptional specificity can be controlled at initiation or termination.,Ancillary factors bind to RNA polymerase as it passes certain sites.The,nut,site consists of two sequences.NusB-S10 join core enzyme as it passes,boxA,.Then NusA and pN protein bind as polymerase passes,boxB,.The presence of pN allows the enzyme to read through the terminator,producing a joint mRNA that contains immediate early sequences joined to delayed early sequences.,RNA polymerase may alternate between initiation-competent and termination-competent forms as sigma and Nus factors alternatively replace one another on the core enzyme.,nusA与核心酶结合，但并不与全酶发生作用。当Sigma因子加入到酶复合体中，它代替了nusA蛋白质，重新组成了全酶。这也就是说RNA酶以准备起始和准备终止的交替形式存在，经历了如图所示的循环。当全酶与启动子结合后，释放了Sigma因子，产生了核心酶来合成RNA，当一个nusA蛋白质辩识了核心酶并与之结合，就产生了酶复合物。当 聚合酶与DNA结合时，nus成分不能被替换。当发生终止时，酶被释放，nus因子或者被释放，或者被sigma因子所取代。,phage regulatory region contains a cluster of trans&cis-factor,Np,Np,Cis-F：P,R,O,R,NuT,R,T,R,P,RE,P,RM,P,L,O,L,NuT,L,T,L1,T,L2,Pint,Trans-F：CI,Cro,CII,N,Q,CIII,int,Xis,4.4.3 Principle of anti-termination inphage,N-p,结合在,NuT,R1,(NuT,L1,),位点,等待,RNApol,Np,使,RNApol,变构并不能与,Rho,因子结合,RNApol,经过,terminator,并发生通读现象,（,read-through,）,pQ,通过在,qut,位点起作用。,qut,序列位于后期转录单位的起点。它的前部分位于起动子之中，后部分在被转录部分的开始。这暗示了,pQ,起作用与,DNA,的识别是联系着的，它作用的机理，至少涉及最初结合复合体，一定与,pN,不同。,pQ,的基本作用是干扰暂停，一旦,pQ,对,RNA,聚合酶起作用，酶在包括,rho-,依赖性和真实终止子在内的所有位点处的暂停将缩短。这样，,pQ,没有直接作用于终止子本身，而是让酶迅速通过终止子，使核心酶和（或）辅助因子没有机会引起终止。,抗终止机制是否多发生于原核生物内？,4.5 Pre-RNA processing,in Eukaryotes,4.5.1,概念,pre-RNA,capping,tailing,splicing,methylation,editing,生物学意义,mature RNA,prevent pre-RNA from digested by RNase,as template,(protein translation),l,interrupted gene,(interrupted RNA),remove introns,(stop codon),4.5.2 pre-RNA capping,l,Cytoplasm Polyhedron Virus(CPV),（胞质多角体病毒）,Jan Furuich(1974),Cap-0 m,7,GpppXpYp-(共有),Cap-1 m,7,GpppX,m,pYp-,Cap-2 m,7,GpppX,m,pY,m,p-,Types&structure of CAP in Eukaryotes,Cap 0,New 3-OH end,Cap 1,Cap 2,m,6,A,the position can be methylated like in Cap1,SAM,SAM,m,7,GpppXmpYp-,m,7,GpppXmpYmp-,SAM;S-Adenosyl-L-methionine,SAH;S-Adenosyl-L-homocysteine,RNA Ns-2-O-methyl transferase,RNA Ns-2-O-methyl transferase,GpppXpYp-,m,7,SAH,(with Cap-1),SAH,(with Cap-2),4.5.3 pre-RNA tailing,概念,A poly(A)tail(50-200)be added,at,-20 Nt,tailing,signal,(AAUAAA)from 3-end of Pre-RNA,Rabbit-globin mRNA,5-CUUUGAAUAAA-poly(A)3,-20,Rabbit-globin mRNA,5-UGGCUAAUAAA-poly(A)3,-20,Man-globin mRNA,5-CUUUGAAUAAA-poly(A)3,-20,Man-globin mRNA,5-UGCCUAAUAAA-poly(A)3,-20,Proceeding of pre-RNA tailing recognizing site,Cap,Cap,特异 内切酶识别AAUAAA及随后的GUGUGUG并在其间酶切，在3端聚合50-200poly(A),GUGUGUG,过程,5 m,7,G-AAUAAA-GUGUGU-3,CPF(cleavage and poly(A)specific factor),PAP(poly(A)polymerase),CSF(cleavage stimulation factor),(stabilize CPF-RNA-PAP complex),CSF,CSF,CPF,PAP,PABP II(poly(A)Binding Protein),Promotion poly(A)elongation,CPF,5 m,7,G-AAUAAA,cleavage,AAAA,AAAAAAAAAAA,CPF,5 m,7,G-AAUAAA,AAAAAAAA A,polymerizing,GUGUGU,A,PAP,CSF,PAP,4.5.4 RNA internal methylation,m,5,C,m,6,A,HNH,HNH,O,O,HNH,HN,CH,3,H,3,C,常见tRNA中的修饰核苷酸,Cmnm,5,U (5-羧甲基氨甲基尿苷),mCm,5,U （5-甲氧基羰甲基尿苷）,Xm,5,s,2,U （5-甲基-2硫代尿苷）,K,2,C （2-赖氨酸胞苷）,Com,5,U （5(2)-羟羧甲基尿苷）,I （Inosine次黄嘌呤）,m,7,G (7-甲基尿苷),m,5,C （5-甲基胞苷）,m,6,A （6-甲基腺苷）,s,2,C （2-硫代胞苷）,(假尿苷）,Um (2-O-甲基尿苷),Q （Queuosine）,Xo,5,U (5-羟基尿苷),OH,OH,NH,CH,2,H,2,N,R,