Size The s70 promoter sequence is betwee

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,Section K: Transcription in Prokaryotes,华中师范大学生科院 杨旭,*,Section K,Transcription in Prokaryotes,K1 Basic Principles of Transcription K2,E. coli,RNA Polymerase K3,E. coli,70,Promoter K4 Transcription, Initiation, Elongation and Termination,Section K: Transcription in Prokaryotes,K1 Basic Principles of Transcription(,转录的基本原则,),Transcription:,an overview,(转录概况),Initiation (,起始,),Elongation (,延伸,),Termination (,终止,),Section K: Transcription in Prokaryotes,Transcription: an overview (,概述,),Definition:,Transcription is the enzymic synthesis of,RNA,on a DNA template.,This is the first stage in the all process of gene expression and finally leads to synthesis of the,protein,encoded by a gene.,Transcription is catalyzed (,催化) by an,RNA polymerase,which requires a,dsDNA template, as well as,the ribo-nucleotides,ATP, GTP, CTP,and,UTP,.,RNA synthesis always occurs in a fixed direction,from the 5- to the 3-end,of the RNA molecule, same as DNA replication.,Section K: Transcription in Prokaryotes,Transcription: an overview (,概述,),Sense strand,: Usually, only one of the two strands of,DNA,becomes transcribed into RNA. One strand is known as the sense strand. The sequence of the RNA is a direct copy of the sequence of the deoxy-nucleotides in the sense strand (with U in place of T).,Antisense strand,(template strand): The other DNA strand is known as the antisense strand. This strand may also be called “template strand” since it is used as the template to which ribo-nucleotides base-pair for the synthesis of the RNA.,Sense strand,template strand,mRNA,Section K: Transcription in Prokaryotes,基因表达的第一步,以D. S. DNA中的一条单链作为转录的模板,在,依赖DNA的RNA聚合酶,的作用下,模板单链 DNA的极性方向为 3 5, 而非模板单链,DNA的极性方向与RNA链合成方向相同，为5 3.,DNA,(文章中书写DNA序列时，仅写非模板序列，可不注明极性方向),3-TACTCAT-5,RNA 5-AUGAGUA-3,5-ATGAGTA-3,Non-template (sense strand),template (antisense strand),概 述,按,A U，C G,配对的原则，合成RNA分子,Section K: Transcription in Prokaryotes,转录复合体,Initiation (,起始,),Steps,:,Bingding,(,结合): The RNA polymerase binds to the dsDNA at a promoter sequence, to initiate RNA synthesis (transcription).,Unwinding,(,解旋): After binding the local DNA unwinds.,Promoters,: They are sequences upstream of the region that codes for protein, and they contain short conserved DNA sequences.,Start site,(,起始位点): The position of the first synthesized base of the RNA is called “start site” and is designated as position +1.,Transcription complex:,The RNA polymerase, when assembled on the DNA template, are often called as transcription complex.,有义链,反义链,5,5,3,3,ATACG,TATGC,P,Promoters,+1,转录区,终止子,5,3,TATGC.,RNA,Section K: Transcription in Prokaryotes,Elongation (,延伸,),Elongation definition,: The process of the RNA polymerase covalently adds ribonucleotides to the 3-end of the growing RNA chain.,Directions,:,The polymerase therefore extends the growing,RNA,chain in a,5,3,direction.,This occurs while the enzyme itself moves in a,3,5,direction along the antisense,DNA,strand (template).,DNA unwinding,and,rewinding,: As the RNA polymerase moves,it locally unwinds the DNA, separating the DNA strands,to expose the template strand 10-17 bp for RNA elongation.,Then the RNA pol rewinds the DNA helix.,Elongation rate,: The,E. coli,RNA polymerase performs this reaction at a rate of around 40 bases per second at 37,C.,Section K: Transcription in Prokaryotes,Termination (,终止,),Terminator,(终止子): The termination of transcription, namely (,即) the dissociation (解体) of the transcription complex and the ending of RNA synthesis, occurs at a specific,DNA,sequence known as terminator.,Hairpin,: The terminators often contain self-complementary regions which can form a hairpin (,发夹) or stem-loop (茎环) secondary structure in the,RNA,product. These cause the RNA pol to cease (终止) transcription.,Accessory factors,: Some terminator sequences require the,rho protein,(,) as an accessory factor (,辅助因子).,Section K: Transcription in Prokaryotes,K2,E. coli,RNA Polymerase,(,大肠杆菌RNA聚合酶),E. coli,RNA Polymerase,(,大肠杆菌RNA聚合酶),Subunit (,亚基,),Subunit (,亚基,), Subunit (,亚基,),Sigma factor (,因子,),Section K: Transcription in Prokaryotes,E. coli,RNA Polymerase,Core enzyme, consisting of 2, 1, 1 and 1 subunits, is responsible for transcription,elongation.,Sigma factor,(), is also required,for correct,transcription,initiation.,After initiation, it will be released from the transcription complex.,Holoenzyme,: The complete enzyme, consisting of the core enzyme and the,factor, is called the holoenzyme. (,例外: T3 and T7 phage has only one,subunit), subunit,: It is 11 kD subunit, but its function is not clear so far.,b,b,s,a,w,a,155 kD,151 kD,70 kD,37 kD,11 kD,Section K: Transcription in Prokaryotes,Subunit,Key Points:,Numbers:,Two identical,a,subunits are present in the core RNA ploymerase enzyme.,Encoding gene,: The subunit is encoded by the,rpoA,gene.,Functions:,In our book,: The,subunits are required for,core enzyme,assembly, but have had no clear transcriptional role assigned to them,?,Our book suggests that the,a,subunit may play a role in promoter recognition.,Other literatures,: Some other literatures suggest that:,One,a,subunit ahead: for the dsDNA unwinding, while,the other,a,subunit latter: for the dsDNA rewinding.,Section K: Transcription in Prokaryotes,Subunit and,b, subunit,b,subunit,: It presents in the core enzyme. It is encoded by the,rpo,B,gene. This subunit is thought to be the,catalytic center,of the RNA pol. The studies suggest the,b,subunit may contain two domains responsible for,initiation,and,elongation,respectively.,Rifampicin,(,利福平) is an inhibitor of RNA polymerase that blocks,initiation,domain;,Strep-tolydigins,(,利迪链霉素) is an inhibitor of,elongation,domain.,b, subunit,: It also presents in the core enzyme. It is encoded by the,rpo,C,gene. The study below suggests that the,b, subunit is responsible for,binding,to the DNA.,Heparin,(,肝素) has shown an ability to bind the,b, subunit of the RNA pol and the transcription will be inhibited by the competition (,竞争) with DNA on binding to the,b, subunit.,Section K: Transcription in Prokaryotes,Structure and function of RNApol (Core) in prok.-I,Enzyme Movement,DNA coding strand (,),Rewinding point (,),Unwinding point (,),RNA binding site,RNA/DNA hybrid (,),DNA template strand,Holo Enzyme,使,DNA,形成,10-17bp,的解链区,Section K: Transcription in Prokaryotes,Structure and function of RNApol (Core) in prok.-II,Enzyme Movement,RNA binding site,RNA/DNA hybrid (,),I,E,I site (Rif,S,),E site (Rif,R,) ;,elongation domain,initiation domain,Section K: Transcription in Prokaryotes,Sigma,(s),factor,Background,:,The most common sigma factor in,E. coli,is,s,70,(70 kDa).,Binding of the,factor converts (将.,转换为) the core RNA pol into the holoenzyme.,Many prokaryotes (including,E. coli,) have multiple,s,factors.,Functions:,The,s,factor has a critical role in,promoter recognition,but is not required for transcription,elongation.,Mechanism:,The,s,factor contributes to promoter recognition by,decreasing,the affinity of the core enzyme for nonspecific DNA sites by a factor of 10,4,and,increasing,affinity for the promoter.,Section K: Transcription in Prokaryotes,K3,E. coli,70,Promoter,(,大肠杆菌,70,启动子),Promoter sequences (,启动子序列),Promoter size (启动子大小),-10 sequence (-10序列),-35 sequence (-35序列),Transcription start site (转录起始点),Promoter efficiency (启动子效率),Section K: Transcription in Prokaryotes,Promoter sequences,Definition,: RNA polymerase binds to specific initiation sites upstream from transcribed sequences. These are called promoter.,s,and,s,70,factor,: Although different promoters are recognized by different,s,factor which interact with the RNA polymerase core enzyme, the most common,s,factor in,E. coli,is,s,70,.,Position number,:,The,start site,generally assigned as position +1;,The,promoter,sequences are assigned as a negative number, reflecting the distance upstream from the start site.,+1,Section K: Transcription in Prokaryotes,Promoter size and positions,Size,: The,s,70,promoter sequence is between 40 and 60 bp long.,Position characteristics,:,-55 to +20:,The region from around -55 to +20 has been shown to be bound by the polymerase;,-20 to +20:,The region from -20 to +20 is strongly protected from nuclease digestion by DNase I (see Topic J4). This suggests that this region is tightly associated with the polymerase which blocks access (,接近) of the nuclease to the DNA,Position -40,: Mutagenesis of promoter sequences showed that sequences up to around position -40 are critical (必须的) for promoter function.,-10 and -35,: Two 6 bp sequences at around positions -10 and -35 have been shown to be particularly (尤其) important for promoter function in,E. coli,.,Section K: Transcription in Prokaryotes,Promoter region (,狭义) including,Sextama Box ;,RNApol.,r,ecognition site (,R,site),TTGACA (Sextama Box),-35 site RNApol. loosely binding site,Pribnow Box ;,TATAAT (pribnow Box),-10 site RNApol. firmly,b,inding site (,B,site),I,nitiation site ;,+1 RNA transcriptional start point (,I,site),A/G,(or called as,start site,),-35,(R),-10,(B),+1,(I),RNA,Section K: Transcription in Prokaryotes,-10 sequence,Definition,: The most conserved sequence in,s,70,promoters is,a 6 bp sequence which is found in the promoters of many,different,E. coli,genes. This sequence is centered at around,the -10 position to the transcription start site (p171 Fig. 1).,TTGACA,1618bp,TATAAT,58bpC,G,T,-35 sequence,-10 sequence,+1,Pribnow box,(,框): The -10 sequence is also referred as the Pribnow box, it was firstly recognized by David Pribnow in 1975.,Consensus (,共有的) sequence,: It is a sequence of,TATAAT,;,Most conserved bases,: The first two bases (TA) and the final T;,Important distance,: This hexamer (,六聚体) is separated by between 5 and 8 bp from the start site. This intervening (插入) base type is not conserved, but the distance is critical.,Function,: The -10 sequence appears to be the sequence at which DNA,unwinding,is initiated by the polymerase (see Topic K4).,+1,Section K: Transcription in Prokaryotes,-35 sequence and start site,-35 sequence,: Upstream regions around position,-35 also have a conserved hexamer (,六聚体) sequence.,Sextama Box,: It was also been found by David Pribnow.,Sextama means hexamer structure.,Consensus,sequence: This has a consensus sequence of,TTG,ACA, which is very conserved in promoters,Most conserved bases: The first three positions of this hexamer (,六聚体) are the most conserved.,Intervening sequence: This sequence is separated by 16-18 bp from the -10 box in 90% of all promoters. The intervening sequence between these conserved elements is not important.,Start site,: The transcription start site is a,purine,in 90% of all genes, and,G,is more common at the transcription start site than,A,. Often, there are,C,and,T,bases on either side of the start site (,CGT,).,TTGACA,1618bp,TATAAT,58bpC,G,T,-35 sequence,-10 sequence,+1,+1,Section K: Transcription in Prokaryotes,Pribnow box,Sextama Box,Start site,Section K: Transcription in Prokaryotes,Promoter efficiency,Efficiency,of different promoter can be defined as follows:,-35 sequence,: constitutes a recognition region which enhances recognition and interaction with the polymerase,s,factor;,-10 sequence,:,is important for DNA unwinding;,Start site,: The sequence around the start site influences initiation.,First 30 bases,: The sequence of the first 30 bases also influences transcription.,Negative supercoiling,: The importance of strand separation in the initiation reaction is shown by the effect of negative supercoiling of the DNA template which generally enhances transcription initiation, because the supercoiled structure requires,less energy,to unwind the DNA.,Section K: Transcription in Prokaryotes,K4 Transcription (,转录,), Initiation (,起始,), Elongation (,延伸,) and Termination (,终止,),Promoter binding (,启动子结合),DNA unwinding (DNA解旋),RNA chain initiation (RNA链起始),RNA chain elongation (RNA链延伸),RNA chain termination (RNA链终止),Rho-dependent termination,(依赖,的转录终止,),Section K: Transcription in Prokaryotes,Promoter binding,Loose binding,(松散结合): The RNA polymerase core enzyme,a,2,bb,w, has a general nonspecific affinity for anywhere DNA. This is referred to as loose binding and, in fact, it is fairly stable.,Mechanism,: When,s,factor is added to the core enzyme to form the holoenzyme, it markedly,reduces,the affinity for,nonspecific,sites on DNA by 20,000 folds.,In addition,s,factor,enhances,holoenzyme binding to,correct promoter-binding,sites 100 times.,Sliding binding,model,: The holoenzyme searches out and binds to promoters in,E. coli,genome extremely rapidly. This process,is,too,fast,to,be achieved by repeated binding and dissociation from DNA, and,is believed to occur by the polymerase,sliding,(,滑动) along the DNA until it reaches the promoter sequence.,Closed complex,(闭链复合物): At the promoter, the RNA pol recognizes the double-stranded -35 and -10 DNA sequences. The initial (,起始) complex of the polymerase with the,base-paired,promoter DNA is referred to as a,closed complex,.,Section K: Transcription in Prokaryotes,DNA unwinding,Negative supercoiling,: In order for the antisense strand to become accessible for base pairing, the DNA duplex must be unwound by the polymerase.,DNA gyrase,: DNA gyrase is responsible for negative supercoiling of the,E. coli,genome and so this may serve as an feedback loop (,反馈调节) for DNA gyrase protein expression.,Tight binding,and,open complex,: 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 (开链复合物),.,Section K: Transcription in Prokaryotes,RNA chain initiation,Features:,1.,First two nt,: The polymerase initially incorporates the first two,nt,and forms a phosphodiester bond between them.,2.,First nine nt,:,The first nine,nt,are added,without enzyme movement,along the DNA.,3.,Other nt,: After the first nine nt is added to the chain, there is a significant probability that the chain will be aborted (,流产).,4.,Clearance,: The minimum time for promoter clearance (清除) is 1-2 seconds, which is a long event relative to other stages,Section K: Transcription in Prokaryotes,Section K: Transcription in Prokaryotes,RNA chain elongation,Ternary,(三联) complex,: When initiation succeeds, the enzyme releases the,s,factor and forms a ternary complex of polymerase-DNA-RNA;,Re-initiation,: Polymerase to progress along the DNA (promoter clearance) allowing re-initiation of transcription from the promoter by a further RNA polymerase holoenzyme.,Transcription bubble,: The region of unwound DNA, which is called the transcription bubble, appears to move along the DNA with the polymerase.,The size of this region of,unwound DNA,remains constant at around 17 bp, and,the 5-end of the RNA forms a,hybrid helix,of about 12 bp with the antisense (template) DNA strand.,This corresponds to just less than one turn of the RNA-DNA helix.,Transcription rate,: The,E. coli,polymerase moves at an average rate of 40 nt per sec, but the rate can vary depending on local DNA sequence.,Section K: Transcription in Prokaryotes,Section K: Transcription in Prokaryotes,RNA chain termination,Stop signal,:,terminator sequence,(stop signal) at the end of the transcription unit. The most common stop signal is an RNA,hairpin,(,发夹) in which the RNA transcript is self-complementary.,Hairpin,:,has a stable structure with a stem and a loop.,Commonly the,stem,is very GC-rich, favoring its base pairing stability.,It seems that the polymerase,pauses,immediately after it has synthesized the hairpin RNA.,U residues,:,The RNA hairpin is often followed by a sequence of four or more,U residues,.,The,U,residues of the RNA pair only weakly with the,A,residues in the antisense DNA strand. The RNA is therefore released from the transcription complex.,Re-annealing,: The antisense strand of DNA then re-anneals with the sense strand and the core enzyme disassociates from the DNA.,N,N N,N C,G C,C G,C G,G C,C G,G C,A U,A U,NNNN UUUU.-OH,Section K: Transcription in Prokaryotes,Rho-dependent termination,Accessory factor,: The RNA polymers can self-terminate at a hairpin structure followed by a stretch of U residues, other terminator sites may not form strong hairpins. They use an accessory factor, the,rho protein,(,),to mediate transcription termination.,Rho protein,:,It is a hexameric (,六聚体) protein that hydrolyzes ATP in the presence of single-stranded RNA.,The protein appears to bind to a stretch of 72 nucleotides in RNA,probably through recognition of a specific structural feature rather than a consensus sequence.,Mechanism,:,Rho moves along the,nascent,RNA towards the transcription complex. There, it enables the RNA polymerase to terminate at rho-dependent transcriptional terminators.,Sometimes, the rho-dependent terminators are hairpin structures, but lack the subsequent stretch of,U residue, which are required for rho-independent termination.,Section K: Transcription in Prokaryotes,Section K: Transcription in Prokaryotes,原 核 生 物 转 录 的 起 始 与 延 伸 过 程,转录,延伸:,30-50 Nt/sec,s,After 9 N t,r,Pausing,1-15 in Term.,RNA,DNA,Pol,Section K: Transcription in Prokaryotes,Thats all for Section K,Section K: Transcription in Prokaryotes,