Size The s70 promoter sequence is betwee

1、 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 TerminationSection K: Transcription in ProkaryotesK1 Basic Principles of Transcription(轉(zhuǎn)錄的基本原則)Transcription: an overview (轉(zhuǎn)錄概況)I

2、nitiation (起始)Elongation (延伸)Termination (終止)Section K: Transcription in ProkaryotesTranscription: 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

3、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: Transcri

4、ption in ProkaryotesTranscription: 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 o

5、f 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 strandtemplate strandmRNASection K: Transcription

6、 in Prokaryotes 基因表達(dá)的第一步 以D. S. DNA中的一條單鏈作為轉(zhuǎn)錄的模板 在依賴(lài)DNA的RNA聚合酶的作用下 模板單鏈 DNA的極性方向?yàn)?3 5, 而非模板單鏈 DNA的極性方向與RNA鏈合成方向相同，為5 3.DNA(文章中書(shū)寫(xiě)DNA序列時(shí)，僅寫(xiě)非模板序列，可不注明極性方向)3-TACTCAT-5RNA 5-AUGAGUA-35-ATGAGTA-3Non-template (sense strand)template (antisense strand)概 述 按A U，C G 配對(duì)的原則，合成RNA分子Section K: Transcription in Pro

7、karyotes轉(zhuǎn)錄復(fù)合體Initiation (起始)Steps: Bingding (結(jié)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 contai

8、n short conserved DNA sequences. Start site (起始位點(diǎn)): 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.有義鏈反義鏈5533ATACGTA

9、TGCPPromoters+1轉(zhuǎn)錄區(qū)終止子53TATGC.RNASection K: Transcription in ProkaryotesElongation (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 53 direction.

10、This occurs while the enzyme itself moves in a 35 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 rewind

11、s the DNA helix. Elongation rate: The E. coli RNA polymerase performs this reaction at a rate of around 40 bases per second at 37C. Section K: Transcription in ProkaryotesTermination (終止)Terminator (終止子): The termination of transcription, namely (即) the dissociation (解體) of the transcription complex

12、 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 (發(fā)夾) or stem-loop (莖環(huán)) secondary structure in the RNA product. These cause the RNA pol to cease (終止) transcription. Acces

13、sory factors: Some terminator sequences require the rho protein () as an accessory factor (輔助因子). Section K: Transcription in ProkaryotesK2 E. coli RNA Polymerase(大腸桿菌RNA聚合酶)E. coli RNA Polymerase (大腸桿菌RNA聚合酶) Subunit (亞基) Subunit (亞基) Subunit (亞基)Sigma factor (因子)Section K: Transcription in Prokary

14、otesE. coli RNA PolymeraseCore 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, con

15、sisting 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.bbsawa155 kD151 kD70 kD37 kD11 kDSection K: Transcription in Prokaryotes SubunitKey Points: Numbers: Two identical a subu

16、nits 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

17、 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 subunitb subunit: It presents in the core enzyme. It is encoded

18、 by the rpoB 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

19、迪鏈霉素) is an inhibitor of elongation domain. b subunit: It also presents in the core enzyme. It is encoded by the rpoC 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 w

20、ill be inhibited by the competition (競(jìng)爭(zhēng)) with DNA on binding to the b subunit. Section K: Transcription in ProkaryotesStructure and function of RNApol (Core) in prok.-IEnzyme MovementDNA coding strand ( )Rewinding point ()Unwinding point ()RNA binding site RNA/DNA hybrid ()DNA template strand Holo E

21、nzyme 使 DNA 形成10-17bp的解鏈區(qū) Section K: Transcription in ProkaryotesStructure and function of RNApol (Core) in prok.-IIEnzyme MovementRNA binding site RNA/DNA hybrid ()IEI site (Rif S) E site (Rif R) ; elongation domaininitiation domainSection K: Transcription in ProkaryotesSigma (s) factorBackground:

22、The most common sigma factor in E. coli is s70 (70 kDa). Binding of the factor converts (將.轉(zhuǎn)換為) 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 transcripti

23、on 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 104 and increasing affinity for the promoter. Section K: Transcription in ProkaryotesK3 E. coli 70 Promoter(大腸桿菌70啟動(dòng)子)Promoter sequences (

24、啟動(dòng)子序列)Promoter size (啟動(dòng)子大小)-10 sequence (-10序列)-35 sequence (-35序列)Transcription start site (轉(zhuǎn)錄起始點(diǎn))Promoter efficiency (啟動(dòng)子效率)Section K: Transcription in ProkaryotesPromoter sequencesDefinition: RNA polymerase binds to specific initiation sites upstream from transcribed sequences. These are called p

25、romoter.s and s70 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 s70. Position number: The start site generally assigned as position +1; The promoter sequences are assigned as a nega

26、tive number, reflecting the distance upstream from the start site. +1Section K: Transcription in ProkaryotesPromoter size and positionsSize: The s70 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 t

27、he 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 DNAPosition -40: Mutagenesis of promoter sequences sh

28、owed that sequences up to around position -40 are critical (必須的) 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 (狹義) inc

29、luding Sextama Box ; RNApol. recognition site (R site)TTGACA (Sextama Box)-35 site RNApol. loosely binding sitePribnow Box ; TATAAT (pribnow Box)-10 site RNApol. firmly binding site (B site)Initiation site ; +1 RNA transcriptional start point (I site)A/G (or called as start site) -35 (R)-10 (B)+1 (I

30、)RNASection K: Transcription in Prokaryotes-10 sequence Definition: The most conserved sequence in s70 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). TTG

31、ACA1618bpTATAAT58bpCGT -35 sequence -10 sequence +1Pribnow 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; Importan

32、t distance: This hexamer (六聚體) 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). +1Section

33、 K: Transcription in Prokaryotes-35 sequence and start site-35 sequence : Upstream regions around position -35 also have a conserved hexamer (六聚體) sequence.Sextama Box: It was also been found by David Pribnow. Sextama means hexamer structure. Consensus sequence: This has a consensus sequence of TTGA

34、CA, which is very conserved in promotersMost conserved bases: The first three positions of this hexamer (六聚體) 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 no

35、t 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). TTGACA1618bpTATAAT58bpCGT -35 sequence -10 sequence +1+1Section K: Transcription

36、in ProkaryotesPribnow boxSextama BoxStart siteSection K: Transcription in ProkaryotesPromoter efficiencyEfficiency 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

37、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

38、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 ProkaryotesK4 Transcription (轉(zhuǎn)錄), Initiation (起始), Elongation (延伸) and Termination (終止)Promoter binding (啟動(dòng)子結(jié)合)DNA

39、 unwinding (DNA解旋)RNA chain initiation (RNA鏈起始)RNA chain elongation (RNA鏈延伸)RNA chain termination (RNA鏈終止)Rho-dependent termination (依賴(lài)的轉(zhuǎn)錄終止)Section K: Transcription in ProkaryotesPromoter bindingLoose binding (松散結(jié)合): The RNA polymerase core enzyme, a2bbw, has a general nonspecific affinity for anyw

40、here 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 p

41、romoter-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 (滑動(dòng)) along the DNA

42、 until it reaches the promoter sequence. Closed complex (閉鏈復(fù)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 Prokary

43、otesDNA unwindingNegative 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 (反饋調(diào)節(jié)) fo

44、r 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 (開(kāi)鏈復(fù)合物). Section K: Transcription in ProkaryotesRNA chain initiationFeatures: 1. First two n

45、t: 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 wil

46、l be aborted (流產(chǎn)).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 ProkaryotesSection K: Transcription in ProkaryotesRNA chain elongationTernary (三聯(lián)) complex: When initiation succeeds, the enzyme rel

47、eases 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 c

48、alled 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 tu

49、rn 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 ProkaryotesSection K: Transcription in ProkaryotesRNA chain terminationStop signal: terminator sequence (s

50、top signal) at the end of the transcription unit. The most common stop signal is an RNA hairpin (發(fā)夾) 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 i