《K原核生物的转录》PPT课件.ppt

SECTION K SECTION K 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 promoter•K4 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 5’3’direction and its sequence corresponds to that of the DNA strand which is known as the sense strand（有义链）.The template of RNA synthesis is the antisense strand. Necessary components: promoter/template, RNA polymerase, NTPs, terminator/template(-) strand is antisense strand. (+) strand is sense strandK1 转录的基本原则转录的基本原则 Initiation: polymerase and promoters Elongation: RNA polymerase Termination: terminatorATACGTATGC+1promoterterminatorTranscribed regionRNADNATranscriptionAntisense strandAUACGStructure of a transcription unitWhat 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）（非专一不稳定的复合物在模板移动）•Slide to find the promoter（封闭的（封闭的“酶酶-启启动子动子”二元复合物）二元复合物）•Unwind the DNA helix （开放的（开放的“酶酶-启动启动子子”二元复合物）二元复合物）•Synthesis of the RNA strand at the start site (initiation site), this position called position +1 （（“酶酶-启动子启动子-rNTP”三三元元复合物）复合物）Transcription bubbleElongation•RNA polymerase adds 核糖核苷酸核糖核苷酸 to the 3’-end and 延伸延伸 the growing RNA chain in the direction of 5’ 3’ （（E. coli: 40 nt/sec)•酶自身沿着反义酶自身沿着反义DNA链（模板）的链（模板）的3’5’方向移动。

方向移动•酶移动时局部解旋，经过后重新形成双酶移动时局部解旋，经过后重新形成双螺旋ElongationTermination•转录复合物的解体转录复合物的解体 and separation of RNA strand from DNA•Occurring at the terminator (often 茎环或茎环或发夹二级发夹二级 structure), some need rho protein as accessory factor.RNA hairpin (发卡发卡) structure5‘ NNNNAAGCGCCGNNNNCCGGCGCUUUUUU -OHNN NN CG•CC•GC•GG•CC•GG•CA•UA•U… NNNN UUUU-OHSteps for RNA transcription2. Requires DNA for activity and is most active with a double-stranded DNA as template. 5’  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4. All RNA polymerases lack 3’  5’ exonuclease activity, and one error usually occurs when 104 to 105 nucleotides are incorporated. 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. 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   ’ for initiation核心酶核心酶:  2   ’  for elongationRNA pol 亚基亚基E. coli polymerase:   subunit •Two identical subunits in the core enzyme •Encoded by the rpoA gene •Required for core protein assembly•May play a role in promoter recognition 1.Encoded by rpoB gene. 2.The catalytic center of the RNA polymerase•Rifampicin (利福平利福平)：：bind to the ββ subunit, and inhibit transcription initiation. Mutation in rpoB gene can result in rifampicin resistance.阻止起始但阻止起始但不影响延伸，治疗不影响延伸，治疗G+的感染和结核病。

的感染和结核病•Streptolydigins(利迪链菌素利迪链菌素)：：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 participate in the catalytic function of the polymerase •Heparin (肝素肝素)：：binds to the  ’ subunit 体体外抑制转录外抑制转录. •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）（与模板结合有关）.E. coli polymerase:  ’ subunit 1.Many 原核生物原核生物 contain multiple   factors 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 1. 1.  factor is critical in promoter recognition, by decreasing 核心酶对非特异序列的亲和核心酶对非特异序列的亲和力力 (104) and increasing the affinity for the corresponding promoter2. 2.  factor is released from the RNA pol after initiation (RNA chain is 8-9 nt)3. 3.  factor 数目明显少于数目明显少于 the other subunits of the RNA pol（（30%））. E. coli polymerase:   factor K3 The E. coli σ70 promoter1. Promoter sequences: -10 sequence and -35 sequence2.Transcription start site3.Promoter efficiencyPromoters: 含有含有RNA pol 特异性结合和转录特异性结合和转录起始所需的保守序列。

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Promoter sequence•位于位于 the start site of transcription (position +1)上游上游, 一般为负数一般为负数•Contains short conserved sequences critical for specific binding of RNA polymerase and transcription initiation•验证主要通过验证主要通过mutations that enhance or diminish the rate of transcription of gene s70 promoter•Consists of a sequence of between 40 and 60 bp•-55 to +20: bound by the polymerase•-20 to +20: tightly associated with the polymerase and protected from nuclease digestion by DNaseΙ•直至直至–40区域对于启动子功能是必须的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 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序列似乎是聚合酶启动DNA解旋的序列解旋的序列-35 sequence 组成增强与聚合酶因组成增强与聚合酶因子相识别和相互作用的识别区子相识别和相互作用的识别区•A conserved 六聚体序列六聚体序列 around 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 promotersTranscription start site •A purine in 90% of all genes•G is more common at position +1 than A•Often, 起始位点两侧为起始位点两侧为C and T (i.e. CGT or CAT)The sequence around the start site influences initiationThe sequences of five E. coli promotersConsensus TTGACATATAATPromoter efficiency (1)•不同启动子与不同基因的转录速率千差万别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 起始位起始位点附近序列点附近序列all influence initiation efficiency.•最初转录最初转录 30 bases 控制控制 the RNA polymerase 离开离开 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. For example, Lac promoter Plac requires cAMP receptor protein (CRP )K4 转录的起始、延伸与终止转录的起始、延伸与终止Promoter binding DNA unwinding RNA chain initiation RNA chain elongation RNA chain termination Rho-dependent termination BindingUnwindingInitiationElongationTerminationPromoter 结合结合•The core enzyme ( 2   ’ ) has nonspecific DNA binding (loose binding, 全酶全酶20000fold less).•The   factor enhances the specificity of the core RNA polymerase ( 2   ’ ) for promoter binding (100x)（全酶结合到正确的位点）（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 promoter DNA(The initial complex of the polymerase with the base-paired promoter DNA) DNA 解旋解旋 •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 DNA results in formation of an open complex with the polymerase and this process is referred to as tight binding RNA chain 起始起始•No primer is needed •Start with a GTP (more common) or ATP •Initially 掺入掺入 first 2 nucleotides，，the first 9 nt are incorporated without polymerase movement along the DNA or σ factor release（未清除启动子）（未清除启动子）•abortive initiations, which are important for the overall rate of transcription，对于聚合酶，对于聚合酶要花多长时间离开启动子并允许另一个聚合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 延伸延伸•σFactor 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 rear•3’ part of RNA 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.antisenseRNA chain 终止终止•Termination: dissociation of RNA > re-annealing of DNA > release of RNA pol•Terminator sequence 终止序列终止序列 (stop signal 停止信号停止信号): •RNA hairpin very common•Accessory rho proteinRNA hairpin 1. RNA 转录物是自身互补的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，有利于解离 A model for ρ-independent termination of transcription in E. coli. The A-U base-pairing is less stable that favors the dissociation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 toward RNA pol complex  stop at the rho-dependent transcription terminator. 注意注意•终止信号的识别发生在新合成的终止信号的识别发生在新合成的RNA连而不是在模板连而不是在模板DNA链中。

链中RNA polymerase/transcription and DNA polymerase/replicationRNA polDNA polTemplatedsDNA is betterss/dsDNARequire primerNoYesInitiationpromoteroriginelongation40 nt/ sec900 bp/secterminatorSynthesized RNA Template DNA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)。