K原核生物的转录

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 s70 promoterK4 转录的起始、延伸与终止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（有义链）.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 转录的基本原则 Initiation: polymerase and promoters Elongation: RNA polymerase Termination: terminatorATACGTAT

3、GC+1promoterterminatorTranscribed regionRNADNATranscriptionAntisense strandAUACGStructure of a transcription unitWhat is a promoter (启动子启动子)?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（位于蛋白质编码区的上游（5端）端）含有短而保守的含有短而保守的DNA序列，不

4、同基因的启序列，不同基因的启动子中序列经常是保守的。动子中序列经常是保守的。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

5、元复合物）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。ElongationTermination 转录复合物的解体 and separation of RNA strand from DNA Occurring at the terminator (often 茎环或发夹二

6、级 structure), some need rho protein as accessory factor.RNA hairpin (发卡发卡) 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、结构，圆柱形孔道旁有一突起，非球形结构，圆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 亚基亚基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.阻止起始但不影响

11、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 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 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 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 核心酶对非特异序列的亲和力 (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 数目明显少于 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. AUACGPromoter sequence 位于 the start site o

16、f 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 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-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-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, 起始位点两侧为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 30 bases 控制 t

21、he RNA polymerase 离开 the promoter, hence influences the rate of the transcription and the overall promoter strength .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 for initiation.

22、For example, Lac promoter Plac requires cAMP receptor protein (CRP )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 BindingUnwindingInitiationElongationTerminationPromoter 结合 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 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. 负超螺旋能增进许多基因的转录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 掺入 first 2 nucleotides，the first 9 nt are incorporated without polymerase movement along the DNA or factor

26、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)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 终止Termination: dissociation of RNA re-annealing of DNA release of RNA polTerminator sequence 终止序列 (stop signal 停止信号): RNA hairpin very commonAccessory rho proteinRNA hair

29、pinRNA 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 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. 注意终止信号的识别发生

31、在新合成的RNA连而不是在模板DNA链中。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