K原核生物的轉(zhuǎn)錄

1、Section K 原核生物的轉(zhuǎn)錄K1 轉(zhuǎn)錄的基本原則K2 Escherichia coli RNA 聚合酶K3 The E. coli s70 promoterK4 轉(zhuǎn)錄的起始、延伸與終止Transcription 是指以a double-stranded DNA 為模板合成 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（有義鏈）.T

2、he template of RNA synthesis is the antisense strand. Necessary components: promoter/template, RNA polymerase, NTPs, terminator/template(-) strand is antisense strand. (+) strand is sense strandK1 轉(zhuǎn)錄的基本原則 Initiation: polymerase and promoters Elongation: RNA polymerase Termination: terminatorATACGTAT

3、GC+1promoterterminatorTranscribed regionRNADNATranscriptionAntisense strandAUACGStructure of a transcription unitWhat is a promoter (啟動(dòng)子啟動(dòng)子)?The sequence of DNA needed for RNA polymerase to bind to the template and accomplish the initiation reaction 位于蛋白質(zhì)編碼區(qū)的上游（位于蛋白質(zhì)編碼區(qū)的上游（5端）端）含有短而保守的含有短而保守的DNA序列，不

4、同基因的啟序列，不同基因的啟動(dòng)子中序列經(jīng)常是保守的。動(dòng)子中序列經(jīng)常是保守的。Initiation Binding of an RNA polymerase to the dsDNA（非專一不穩(wěn)定的復(fù)合物在模板移動(dòng)）Slide to find the promoter（封閉的“酶-啟動(dòng)子”二元復(fù)合物）Unwind the DNA helix （開放的“酶-啟動(dòng)子”二元復(fù)合物）Synthesis of the RNA strand at the start site (initiation site), this position called position +1 （“酶-啟動(dòng)子-rNTP”三

5、元復(fù)合物）Transcription bubbleElongationRNA 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)酶自身沿著反義DNA鏈（模板）的35方向移動(dòng)。酶移動(dòng)時(shí)局部解旋，經(jīng)過后重新形成雙螺旋。ElongationTermination 轉(zhuǎn)錄復(fù)合物的解體 and separation of RNA strand from DNA Occurring at the terminator (often 莖環(huán)或發(fā)夾二

6、級(jí) structure), some need rho protein as accessory factor.RNA hairpin (發(fā)卡發(fā)卡) structure5 NNNNAAGCGCCGNNNNCCGGCGCUUUUUU -OHNN NN CGCCGCGGCCGGCAUAU NNNN UUUU-OHSteps for RNA transcription2. Requires DNA for activity and is most active with a double-stranded DNA as template. 5 3 synthesis(NMP)n + NTP (NMP

7、)n+1 + PPiRNA polymerase: synthesis of RNA strand from DNA template.1. Requires no primer for polymerization3. Require Mg2+ for RNA synthesis activity4. All RNA polymerases lack 3 5 exonuclease activity, and one error usually occurs when 104 to 105 nucleotides are incorporated. 5. usually are multis

8、ubunit 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. K2 E. coli RNA polymeraseBoth 起始起始 & 延伸延伸起始起始 only36.5 KD36.5 KD151 KD155 KD11 KD70 KD465kd RNA synthesis rate: 40 nt per second at 37oC 非球形

9、結(jié)構(gòu)，圓柱形孔道旁有一突起，非球形結(jié)構(gòu)，圓柱形孔道旁有一突起，孔道大小表明孔道大小表明the enzyme complex can bind directly to 16 bp of DNA. The whole polymerase binds over 60 bp.E. coli RNA polymerase:全酶全酶: 2 ss for initiation核心酶核心酶: 2 for elongationRNA pol 亞基亞基E. coli polymerase: subunit Two identical subunits in the core enzyme Encoded by

10、the rpoA gene Required for core protein assemblyMay play a role in promoter recognition 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 resistance.阻止起始但不影響

11、延伸，治療G+的感染和結(jié)核病。Streptolydigins(利迪鏈菌素)：resistant mutations are mapped to rpoB gene as well. Inhibits transcription elongation but not initiation. 3.3. subunit may contain two domains responsible for 轉(zhuǎn)錄起始和延伸。E. coli polymerase: subunit 1.Encoded by the rpoC gene .2.Binds two Zn 2+ ions and may partici

12、pate in the catalytic function of the polymerase Heparin (肝素)：binds to the subunit 體外抑制轉(zhuǎn)錄. Heparin competes with DNA for binding to the polymerase.3. subunit may be responsible for binding to the template DNA （與模板結(jié)合有關(guān)）.E. coli polymerase: subunit 1. Many 原核生物 contain multiple s s factors to recogniz

13、e different promoters. The most common s s factor in E. coli is s s70.2. Binding of the s s factor converts the core RNA pol into the holoenzyme.E. coli polymerase: s factor 1.1.s s factor is critical in promoter recognition, by decreasing 核心酶對非特異序列的親和力 (104) and increasing the affinity for the corr

14、esponding promoter2.2.s s factor is released from the RNA pol after initiation (RNA chain is 8-9 nt)3.3.s s factor 數(shù)目明顯少于 the other subunits of the RNA pol（30%）. E. coli polymerase: s factor K3 The E. coli 70 promoter1. Promoter sequences: -10 sequence and -35 sequence2.Transcription start site3.Pro

15、moter efficiencyPromoters: 含有RNA pol 特異性結(jié)合和轉(zhuǎn)錄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. AUACGPromoter sequence 位于 the start site o

16、f transcription (position +1)上游, 一般為負(fù)數(shù) Contains short conserved sequences critical for specific binding of RNA polymerase and transcription initiation 驗(yàn)證主要通過mutations that enhance or diminish the rate of transcription of gene s s70 promoter Consists of a sequence of between 40 and 60 bp -55 to +20:

17、bound by the polymerase -20 to +20: tightly associated with the polymerase and protected from nuclease digestion by DNase 直至40區(qū)域?qū)τ趩?dòng)子功能是必須的 -10 and 35 sequence: particularly important for promoter function-5-8 bp- GC T ATTGACATATAAT-16-18 bp-+1-35 sequence-10 sequence-10 sequence (Pribnow box) 6 bp

18、sequence which is centered at around the 10 position (Pribnow, 1975). A consensus sequence of TATAAT The first two bases(TA) and the final T are most highly conserved among other E. coli promoters 六聚體距離轉(zhuǎn)錄起始位點(diǎn)5 to 8 bp -10序列似乎是聚合酶啟動(dòng)DNA解旋的序列-35 sequence 組成增強(qiáng)與聚合酶因子相識(shí)別和相互作用的識(shí)別區(qū) A conserved 六聚體序列 around

19、position 35 A consensus sequence of TTGACA The 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 promotersTranscription start site A purine in 90% of all genes G is more common at position +1 than A Often, 起始位點(diǎn)兩側(cè)為C and T (

20、i.e. CGT or CAT)The sequence around the start site influences initiationThe sequences of five E. coli promotersConsensus TTGACATATAATPromoter efficiency (1) 不同啟動(dòng)子與不同基因的轉(zhuǎn)錄速率千差萬別vary by up to 1000-fold . The 35 sequence, -10 sequence, and 起始位點(diǎn)附近序列all influence initiation efficiency. 最初轉(zhuǎn)錄 30 bases 控制 t

21、he RNA polymerase 離開 the promoter, hence influences the rate of the transcription and the overall promoter strength .Promoter efficiency (2) DNA模板的負(fù)超螺旋可增強(qiáng)轉(zhuǎn)錄起始 Some promoter sequence are not strong enough to initiate transcription under normal condition, activating factor is required for initiation.

22、For example, Lac promoter Plac requires cAMP receptor protein (CRP )K4 轉(zhuǎn)錄的起始、延伸與終止Promoter binding DNA unwinding RNA chain initiation RNA chain elongation RNA chain termination Rho-dependent termination BindingUnwindingInitiationElongationTerminationPromoter 結(jié)合 The core enzyme ( 2 ) has nonspecific

23、DNA binding (loose binding, 全酶20000fold less). The s s factor enhances the specificity of the core RNA polymerase ( 2 ) for promoter binding (100 x)（全酶結(jié)合到正確的位點(diǎn)） The polymerase finds the promoter 35 and 10 sequences by sliding along the DNA extremely rapidly and forming a closed complex with the prom

24、oter DNA(The initial complex of the polymerase with the base-paired promoter DNA) DNA 解旋 Necessary to unwind the DNA so that the antisense strand to become accessible for base pairing, carried out by the polymerase. 負(fù)超螺旋能增進(jìn)許多基因的轉(zhuǎn)錄but not all facilitating by unwinding . The initial unwinding of the D

25、NA results in formation of an open complex with the polymerase and this process is referred to as tight binding RNA chain 起始 No primer is needed Start with a GTP (more common) or ATP Initially 摻入 first 2 nucleotides，the first 9 nt are incorporated without polymerase movement along the DNA or factor

26、release（未清除啟動(dòng)子） abortive initiations, which are important for the overall rate of transcription，對于聚合酶要花多長時(shí)間離開啟動(dòng)子并允許另一個(gè)聚合酶起始新一輪轉(zhuǎn)錄重要作用 The minimum time for promoter clearance is 1-2 seconds (a relatively long event)RNA chain RNA chain 延伸 Factor is released to form a ternary complex of the pol-DNA-RNA

27、(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 rear 3 part of RNA forms hybrid helix (ca. 12bp) with antisense DNA

28、 strand. The E. coli polymerase moves at an average rate of 40 nt per sec, depending on the local DNA sequence.antisenseRNA chain 終止Termination: dissociation of RNA re-annealing of DNA release of RNA polTerminator sequence 終止序列 (stop signal 停止信號(hào)): RNA hairpin very commonAccessory rho proteinRNA hair

29、pinRNA hairpin 1. RNA 轉(zhuǎn)錄物是自身互補(bǔ)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，有利于解離 A model for -independent termination of transcription in E. coli. The A-U base-pai

30、ring is less stable that favors the dissociation2. 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 toward RNA pol complex stop at the rho-dependent transcription terminator. 注意終止信號(hào)的識(shí)別發(fā)生

31、在新合成的RNA連而不是在模板DNA鏈中。RNA polymerase/transcription and DNA polymerase/replicationRNA polDNA polTemplatedsDNA is betterss/dsDNARequire primerNoYesInitiationpromoteroriginelongation40 nt/ sec900 bp/secterminatorSynthesized RNA Template DNASummary:1. General features of RNA pol.2. E. coli RNA pol: holoe

32、nzyme ( 2 ss) and core enzyme ( 2 ). Role of sigma factor.3. E. coli s s70 promoter: -35 sequence (TTGACA), -10 (TATAAT), & +1 (purine, G A)4. Transcription: initiation, elongation and termination (hairpin and rho factor)ATACGTATGC+1promoterterminatorTranscribed regionRNADNATranscriptionAntisense strandAUACGStructure of a transcription unit