K原核生物的转录.pptx

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1、Section K 原核生物的转录K1 转录的基本原则K2 Escherichia coli RNA 聚合酶K3 The E.coli 70 promoterK4 转录的起始、延伸与终止第1页/共54页Transcription 是指以a double-stranded DNA 为模板合成 a single-stranded RNA.RNA synthesis occurs in the 53direction and its sequence corresponds to that of the DNA strand which is known as the sense strand（有义

2、链）.The template of RNA synthesis is the antisense strand.Necessary components:promoter/template,RNA polymerase,NTPs,terminator/template第2页/共54页(-)strand is antisense strand.(+)strand is sense strand第3页/共54页K1 转录的基本原则 Initiation:polymerase and promoters Elongation:RNA polymerase Termination:terminato

3、r第4页/共54页ATACGTATGC+1promoterterminatorTranscribed regionRNADNATranscriptionAntisense strandAUACGStructure of a transcription unit第5页/共54页What is a promoter(启动子)?The sequence of DNA needed for RNA polymerase to bind to the template and accomplish the initiation reaction 位于蛋白质编码区的上游（5端）含有短而保守的DNA序列，不

4、同基因的启动子中序列经常是保守的。第6页/共54页Initiation Binding of an RNA polymerase to the dsDNA（非专一不稳定的复合物在模板移动）Slide to find the promoter（封闭的“酶-启动子”二元复合物）Unwind the DNA helix（开放的“酶-启动子”二元复合物）Synthesis of the RNA strand at the start site(initiation site),this position called position+1（“酶-启动子-rNTP”三元复合物）第7页/共54页Trans

5、cription bubble第8页/共54页ElongationRNA polymerase adds 核糖核苷酸 to the 3-end and 延伸 the growing RNA chain in the direction of 5 3（E.coli:40 nt/sec)酶自身沿着反义DNA链（模板）的35方向移动。酶移动时局部解旋，经过后重新形成双螺旋。第9页/共54页Elongation第10页/共54页Termination转录复合物的解体 and separation of RNA strand from DNAOccurring at the terminator(oft

6、en 茎环或发夹二级 structure),some need rho protein as accessory factor.第11页/共54页RNA hairpin(发卡)structure5 NNNNAAGCGCCGNNNNCCGGCGCUUUUUU-OHNN NN CGCCGCGGCCGGCAUAU NNNN UUUU-OH第12页/共54页Steps for RNA transcription第13页/共54页2.Requires DNA for activity and is most active with a double-stranded DNA as template.5

7、3 synthesis(NMP)n+NTP (NMP)n+1+PPiRNA polymerase:synthesis of RNA strand from DNA template.1.Requires no primer for polymerization3.Require Mg2+for RNA synthesis activity第14页/共54页4.All RNA polymerases lack 3 5 exonuclease activity,and one error usually occurs when 104 to 105 nucleotides are incorpor

8、ated.5.usually are multisubunit enzyme,but not always.6.Different from organism to organism7.E.coli has a single DNA-directed RNA polymerase that synthesizes all types of RNA.第15页/共54页K2 E.coli RNA polymeraseBoth 起始&延伸起始 only36.5 KD36.5 KD151 KD155 KD11 KD70 KD465kd第16页/共54页 RNA synthesis rate:40 nt

9、 per second at 37oC 非球形结构，圆柱形孔道旁有一突起，孔道大小表明the enzyme complex can bind directly to 16 bp of DNA.The whole polymerase binds over 60 bp.第17页/共54页E.coli RNA polymerase:全酶:2 for initiation核心酶:2 for elongation第18页/共54页RNA pol 亚基第19页/共54页E.coli polymerase:subunit Two identical subunits in the core enzyme

10、Encoded by the rpoA gene Required for core protein assemblyMay play a role in promoter recognition 第20页/共54页1.Encoded by rpoB gene.2.The catalytic center of the RNA polymeraseRifampicin(利福平)：bind to the subunit,and inhibit transcription initiation.Mutation in rpoB gene can result in rifampicin resis

11、tance.阻止起始但不影响延伸，治疗G+的感染和结核病。Streptolydigins(利迪链菌素)：resistant mutations are mapped to rpoB gene as well.Inhibits transcription elongation but not initiation.3.3.subunit may contain two domains responsible for 转录起始和延伸。E.coli polymerase:subunit 第21页/共54页1.Encoded by the rpoC gene.2.Binds two Zn 2+ions

12、 and may participate in the catalytic function of the polymerase Heparin(肝素)：binds to the subunit 体外抑制转录.Heparin competes with DNA for binding to the polymerase.3.subunit may be responsible for binding to the template DNA（与模板结合有关）.E.coli polymerase:subunit 第22页/共54页1.Many 原核生物 contain multiple facto

13、rs to recognize different promoters.The most common factor in E.coli is 70.2.Binding of the factor converts the core RNA pol into the holoenzyme.E.coli polymerase:factor 第23页/共54页1.1.factor is critical in promoter recognition,by decreasing 核心酶对非特异序列的亲和力(104)and increasing the affinity for the corres

14、ponding promoter2.2.factor is released from the RNA pol after initiation(RNA chain is 8-9 nt)3.3.factor 数目明显少于 the other subunits of the RNA pol（30%）.E.coli polymerase:factor 第24页/共54页K3 The E.coli 70 promoter1.Promoter sequences:-10 sequence and-35 sequence2.Transcription start site3.Promoter effic

15、iency第25页/共54页Promoters:含有RNA pol 特异性结合和转录起始所需的保守序列。ATACGTATGC+1promoterterminatorTranscribed regionRNADNATranscriptionAntisense strandDifferent promoters result in differing efficiencies of transcription initiation,which in turn regulate transcription.AUACG第26页/共54页Promoter sequence位于 the start sit

16、e of transcription(position+1)上游,一般为负数Contains short conserved sequences critical for specific binding of RNA polymerase and transcription initiation验证主要通过mutations that enhance or diminish the rate of transcription of gene 第27页/共54页第28页/共54页 70 promoterConsists of a sequence of between 40 and 60 bp

17、-55 to+20:bound by the polymerase-20 to+20:tightly associated with the polymerase and protected from nuclease digestion by DNase直至40区域对于启动子功能是必须的-10 and 35 sequence:particularly important for promoter function-5-8 bp-GC T ATTGACATATAAT-16-18 bp-+1-35 sequence-10 sequence第29页/共54页-10 sequence(Pribnow

18、 box)6 bp sequence which is centered at around the 10 position(Pribnow,1975).A consensus sequence of TATAATThe first two bases(TA)and the final T are most highly conserved among other E.coli promoters六聚体距离转录起始位点5 to 8 bp-10序列似乎是聚合酶启动DNA解旋的序列第30页/共54页-35 sequence 组成增强与聚合酶因子相识别和相互作用的识别区A conserved 六聚体

19、序列 around position 35A consensus sequence of TTGACAThe first three positions(TTG)are the most conserved among E.coli promoters.Separated by 16-18 bp from the 10 box in 90%of all promoters第31页/共54页Transcription start site A purine in 90%of all genesG is more common at position+1 than AOften,起始位点两侧为C

20、and T(i.e.CGT or CAT)The sequence around the start site influences initiation第32页/共54页The sequences of five E.coli promotersConsensusTTGACATATAAT第33页/共54页Promoter efficiency(1)不同启动子与不同基因的转录速率千差万别vary by up to 1000-fold.The 35 sequence,-10 sequence,and 起始位点附近序列all influence initiation efficiency.最初转录

21、 30 bases 控制 the RNA polymerase 离开 the promoter,hence influences the rate of the transcription and the overall promoter strength.第34页/共54页Promoter efficiency(2)DNA模板的负超螺旋可增强转录起始Some promoter sequence are not strong enough to initiate transcription under normal condition,activating factor is required

22、 for initiation.For example,Lac promoter Plac requires cAMP receptor protein(CRP)第35页/共54页K4 转录的起始、延伸与终止Promoter binding DNA unwinding RNA chain initiation RNA chain elongation RNA chain termination Rho-dependent termination 第36页/共54页Binding第37页/共54页UnwindingInitiationElongationTermination第38页/共54页P

23、romoter 结合The core enzyme(2 )has nonspecific DNA binding(loose binding,全酶20000fold less).The factor enhances the specificity of the core RNA polymerase(2 )for promoter binding(100 x)（全酶结合到正确的位点）The polymerase finds the promoter 35 and 10 sequences by sliding along the DNA extremely rapidly and formi

24、ng a closed complex with the promoter DNA(The initial complex of the polymerase with the base-paired promoter DNA)第39页/共54页 DNA 解旋 Necessary to unwind the DNA so that the antisense strand to become accessible for base pairing,carried out by the polymerase.负超螺旋能增进许多基因的转录but not all facilitating by un

25、winding.The initial unwinding of the DNA results in formation of an open complex with the polymerase and this process is referred to as tight binding 第40页/共54页RNA chain 起始No primer is needed Start with a GTP(more common)or ATP Initially 掺入 first 2 nucleotides，the first 9 nt are incorporated without

26、polymerase movement along the DNA or factor release（未清除启动子）abortive initiations,which are important for the overall rate of transcription，对于聚合酶要花多长时间离开启动子并允许另一个聚合酶起始新一轮转录重要作用The minimum time for promoter clearance is 1-2 seconds(a relatively long event)第41页/共54页第42页/共54页RNA chain RNA chain 延伸Factor

27、is released to form a ternary complex of the pol-DNA-RNA(newly synthesized),causing the polymerase to progress along the DNA(promoter clearance)Transcription bubble(unwound DNA region,17 bp)moves along the DNA with RNA polymerase which unwinds DNA at the front and rewinds it at the rear3 part of RNA

28、 forms hybrid helix(ca.12bp)with antisense DNA strand.The E.coli polymerase moves at an average rate of 40 nt per sec,depending on the local DNA sequence.第43页/共54页antisense第44页/共54页第45页/共54页RNA chain 终止Termination:dissociation of RNA re-annealing of DNA release of RNA polTerminator sequence 终止序列(sto

29、p signal 停止信号):RNA hairpin very commonAccessory rho protein第46页/共54页RNA hairpinRNA hairpin 1.RNA 转录物是自身互补的2.GC-rich favouring the base pairing stability and causing the polymerase to pause3.Followed by a stretch of four or more Us which result in weak RNA-antisense DNA strand binding，有利于解离第47页/共54页

30、A model for-independent termination of transcription in E.coli.The A-U base-pairing is less stable that favors the dissociation第48页/共54页2.Rho-dependent Termination.lNo U stretch at the 3 end of the RNAlRho protein(hexameric protein)binds to certain RNA structure(72bp)moves along the nascent RNA towa

31、rd RNA pol complex stop at the rho-dependent transcription terminator.第49页/共54页第50页/共54页注意终止信号的识别发生在新合成的RNA连而不是在模板DNA链中。第51页/共54页RNA polymerase/transcription and DNA polymerase/replicationRNA polDNA polTemplatedsDNA is betterss/dsDNARequire primerNoYesInitiationpromoteroriginelongation40 nt/sec900 b

32、p/secterminatorSynthesized RNA Template DNA第52页/共54页Summary:1.General features of RNA pol.2.E.coli RNA pol:holoenzyme(2)and core enzyme(2).Role of sigma factor.3.E.coli 70 promoter:-35 sequence(TTGACA),-10(TATAAT),&+1(purine,G A)4.Transcription:initiation,elongation and termination(hairpin and rho factor)第53页/共54页感谢您的观看！第54页/共54页