分子生物学之原核生物的转录

1、分子生物学之原核生物的转录第1页，共62页，2022年，5月20日，9点58分，星期一Molecular Biology CourseSection B: Transcription in prokaryotesPart I: The Central Dogma 第2页，共62页，2022年，5月20日，9点58分，星期一B1 Basic principles of transcriptionB2 Escherichia coli RNA polymeraseB3 The E. coli s70 promoterB4 transcription process. An overview, th

2、e process of RNA synthesis ( initiation, elongation, termination)Properties, a subunit, b subunit, b subunit, sigma (s) factorPromoter, s70 size, -10 sequence, -35 sequence, transcription start site, promoter efficiencyPromoter binding, unwinding, RNA chain initiation, elongation, termination (r fac

3、tor)Section B: Transcription in prokaryotes第3页，共62页，2022年，5月20日，9点58分，星期一Molecular Biology CourseB1: Basic principles of transcriptionTranscription: an overview (comparison with replication)The process of RNA synthesis: initiation, elongation, termination第4页，共62页，2022年，5月20日，9点58分，星期一Molecular Biolo

4、gy CourseB1-1: Transcription: an overview第5页，共62页，2022年，5月20日，9点58分，星期一asymmetric transcription第6页，共62页，2022年，5月20日，9点58分，星期一Key terms defined in this section (Gene VII)Coding strand of DNA has the same sequence as mRNA.Downstream identifies sequences proceeding further in the direction of expressio

5、n; for example, the coding region is downstream of the initiation codon.Gene XPrimary transcriptm7GpppAAAAAn+1upstreamdownstreammRNA第7页，共62页，2022年，5月20日，9点58分，星期一Upstream identifies sequences proceeding in the opposite direction from expression; for example, the bacterial promoter is upstream from t

6、he transcription unit, the initiation codon is upstream of the coding region.Transcription unit is the distance between sites of initiation and termination by RNA polymerase; may include more than one gene.Promoter is a region of DNA involved in binding of RNA polymerase to initiate transcription 第8

7、页，共62页，2022年，5月20日，9点58分，星期一RNA Terminator is a sequence of DNA, represented at the end of the transcript, that causes RNA polymerase to terminate transcription. RNA polymerases are enzymes that synthesize RNA using a DNA template (formally described as DNA-dependent RNA polymerases).Primary transcr

8、ipt is the original unmodified RNA product corresponding to a transcription unit.第9页，共62页，2022年，5月20日，9点58分，星期一Replication: synthesis of two DNA molecules using both parental DNA strands as templates. Duplication of a DNA molecule. 1 DNA molecule 2 DNA moleculesTranscription: synthesis of one RNA mo

9、lecule using one of the two DNA strands as a template.1 DNA molecule 1 RNA moleculeB1: Basic principles of transcription第10页，共62页，2022年，5月20日，9点58分，星期一Replication-synthesis of the leading strandthe same direction as the replication fork movesReview of replication第11页，共62页，2022年，5月20日，9点58分，星期一Replic

10、ation- Synthesis of the Okazaki fragmentsOpposite to the replication fork movementReview of replication第12页，共62页，2022年，5月20日，9点58分，星期一Coupling the synthesis of leading and lagging strands with a dimeric DNA pol III (E. coli)第13页，共62页，2022年，5月20日，9点58分，星期一TranscriptionB1: Basic principles of transcri

11、ption第14页，共62页，2022年，5月20日，9点58分，星期一RNA synthesis occurs in the 53 direction and its sequence corresponds to the sense strand (coding strand).The template of RNA synthesis is the antisense strand (template strand). Phosphodiester bonds: same as in DNANecessary components: RNA polymerase, transcripti

12、on factors, rNTPs, promoter & terminator/templateB1: Basic principles of transcription第15页，共62页，2022年，5月20日，9点58分，星期一B1-2: The process of RNA synthesisinitiationelongationterminationB1: Basic principles of transcription第16页，共62页，2022年，5月20日，9点58分，星期一Flowchart of RNA synthesisBack 1, 2第17页，共62页，2022年

13、，5月20日，9点58分，星期一B1: Basic principles of transcriptionPromoterTerminatorTranscribed regionSense strandAntisense strandDNARNATranscription+1Fig. 2. Structure of a typical transcription unitIs transcribed region equal to coding region? Why?第18页，共62页，2022年，5月20日，9点58分，星期一B3-1: PromoterThe specific short

14、 conserved DNA sequences:upstream from the transcribed sequence, and thus assigned a negative number (location) required for specific binding of RNA Pol. and transcription initiation (function) Were first characterized through mutations that enhance or diminish the rate of transcription of gene 第19页

15、，共62页，2022年，5月20日，9点58分，星期一开始转录T T G A C AA A C T G T-35 区(Pribnow box)T A T A A T Pu A T A T T A Py-10 区1-30-5010-10-40-205 3 3 5 PromoterRNA-pol辨认位点(recognition site) 55RNA聚合酶保护区结构基因33第20页，共62页，2022年，5月20日，9点58分，星期一-10 sequence (Pribonow box)The most conserved sequence in s70 promoters at which DN

16、A unwinding is initiated by RNA Pol.A 6 bp sequence which is centered at around the 10 position, and is found in the promoters of many different E. coli gene. The consensus sequence is TATAAT. The first two bases (TA) and the final T are most highly conserved.This hexamer is separated by between 5 a

17、nd 8 bp from position +1, and the distance is critical.B3: The E. coli s70 promoter第21页，共62页，2022年，5月20日，9点58分，星期一-35 sequence: enhances recognition and interaction with the polymerase s factor A conserved hexamer sequence around position 35A consensus sequence of TTGACAThe first three positions (TT

18、G) are the most conserved among E. coli promoters.Separated by 16-18 bp from the 10 box in 90% of all promotersB3: The E. coli s70 promoter第22页，共62页，2022年，5月20日，9点58分，星期一Footprinting is a technique derived from principles used in DNA sequencing. It is used to identify the specific DNA sequences that

19、 are bound by a particular protein. RNA Polymerase Leaves Its FootPrint on a PromoterSupplemental material第23页，共62页，2022年，5月20日，9点58分，星期一Supplemental materialFootprinting第24页，共62页，2022年，5月20日，9点58分，星期一Supplemental materialFootprinting第25页，共62页，2022年，5月20日，9点58分，星期一RNA聚合酶保护法第26页，共62页，2022年，5月20日，9点58

20、分，星期一B3-5: Transcription start siteB3: The E. coli s70 promoterIs a purine in 90% of all geneG is more common at position +1 than AThere are usually a C and T on either side of the start nucleotide (i.e. CGT or CAT)第27页，共62页，2022年，5月20日，9点58分，星期一The sequences of five E. coli promotersB3: The E. coli

21、 s70 promoter第28页，共62页，2022年，5月20日，9点58分，星期一Molecular Biology CourseB2 Escherichia coli RNA polymeraseE. coli RNA polymerase a subunitb subunitb subunitsigma (s) factor第29页，共62页，2022年，5月20日，9点58分，星期一B2: E. coli RNA polymeraseB2-1 E. coli RNA polymeraseSynthesis of single-stranded RNA from DNA templa

22、te.第30页，共62页，2022年，5月20日，9点58分，星期一Requires no primer for polymerizationRequires DNA for activity and is most active with a double-stranded DNA as template.5 3 synthesisRequire Mg2+ for RNA synthesis activitylacks 3 5 exonuclease activity, and the error rate of nucleotides incorporation is 10-4 to 10

23、-5. Is this accuracy good enough for gene expression?6. usually are multisubunit enzyme.B2: E. coli RNA polymeraseRNA polymerase(NMP)n + NTP (NMP)n+1 + PPi第31页，共62页，2022年，5月20日，9点58分，星期一E. coli polymeraseE. coli has a single DNA-directed RNA polymerase that synthesizes all types of RNA. One of the l

24、argest enzyme in the cellsConsists of at least 5 subunits in the holoenzyme, 2 alpha (a), and 1 of beta (b), beta prime (b), omega (w) and sigma (s) subunits Shaped as a cylindrical channel that can bind directly to 16 bp of DNA. The whole polymerase binds over 60 bp.RNA synthesis rate: 40 nt per se

25、cond at 37oCB2: E. coli RNA polymerase第32页，共62页，2022年，5月20日，9点58分，星期一E. coli RNA polymeraseBoth initiation & elongationInitiation only36.5 KD36.5 KD151 KD155 KD11 KD70 KDB2: E. coli RNA polymerase第33页，共62页，2022年，5月20日，9点58分，星期一B2-1: Sigma (s) factorB2: E. coli RNA polymerase第34页，共62页，2022年，5月20日，9点5

26、8分，星期一Many prokaryotes contain multiple s factors to recognize different promoters. The most common s factor in E. coli is s70.Binding of the s factor converts the core RNA pol into the holoenzyme.s factor is critical in promoter recognition, by decreasing the affinity of the core enzyme for non-spe

27、cific DNA sites (104) and increasing the affinity for the corresponding promoters factor is released from the RNA pol after initiation (RNA chain is 8-9 nt)Less amount of s factor is required in cells than that of the other subunits of the RNA pol. 第35页，共62页，2022年，5月20日，9点58分，星期一 B2-2: a subunitB2:

28、E. coli RNA polymerase第36页，共62页，2022年，5月20日，9点58分，星期一E. coli polymerase: a subunit Two identical subunits in the core enzyme Encoded by the rpoA gene Required for assembly of the core enzymePlays a role in promoter recognition. Experiment: When phage T4 infects E. coli, the subunit is modified by AD

29、P-ribosylation of an arginine. The modification is associated with a reduced affinity for the promoters formerly recognized by the holoenzyme. plays a role in the interaction of RNA polymerase with some regulatory factorsB2: E. coli RNA polymerase第37页，共62页，2022年，5月20日，9点58分，星期一B2-3&4: b and b subuni

30、tB2: E. coli RNA polymerase第38页，共62页，2022年，5月20日，9点58分，星期一b is encoded by rpoB gene, and b is encoded by rpoC gene . Make up the catalytic center of the RNA polymeraseTheir sequences are related to those of the largest subunits of eukaryotic RNA polymerases, suggesting that there are common features

31、 to the actions of all RNA polymerases. The b subunit can be crosslinked to the template DNA, the product RNA, and the substrate ribonucleotides; mutations in rpoB affect all stages of transcription. Mutations in rpoC show that b also is involved at all stages.第39页，共62页，2022年，5月20日，9点58分，星期一b subuni

32、t may contain two domains responsible for transcription initiation and elongationRifampicin (利福平)：has been shown to bind to the subunit, and inhibit transcription initiation by prokaryotic RNA pol. Mutation in rpoB gene can result in rifampicin resistance.Streptolydigins(利迪链菌素)：resistant mutations a

33、re mapped to rpoB gene as well. Inhibits transcription elongation but not initiation. B2: E. coli RNA polymerase第40页，共62页，2022年，5月20日，9点58分，星期一b subunit Binds two Zn 2+ ions and may participate in the catalytic function of the polymerase Hyparin (肝素)：binds to the b subunit and inhibits transcription

34、 in vitro. Hyparin competes with DNA for binding to the polymerase.2.b subunit may be responsible for binding to the template DNA .B2: E. coli RNA polymerase第41页，共62页，2022年，5月20日，9点58分，星期一B3-6: promoter efficiencyB3: The E. coli s70 promoterThere is considerable variation in sequence between differe

35、nt promoters, and the transcription efficiency can vary by up to 1000-fold .第42页，共62页，2022年，5月20日，9点58分，星期一Some promoter sequence are not sufficiently similar to the consensus sequence to be strongly transcribed under normal condition, thus activating factor is required for efficient initiation. Exa

36、mple: Lac promoter P lac requires activating protein, cAMP receptor protein (CRP ), to bind to a site on the DNA close to the promoter sequence in order to enhance polymerase binding and transcription initiation.Weak promoters and activating factor第43页，共62页，2022年，5月20日，9点58分，星期一Molecular Biology Cou

37、rseB4 Transcription process Promoter bindingDNA unwindingRNA chain initiationRNA chain elongationRNA chain termination (r factor)第44页，共62页，2022年，5月20日，9点58分，星期一Promoter bindingB4 Transcription processThe searching process is extremely rapidlyClosed complex: the initial complex of the polymerase with

38、 the base-paired promoter DNA)and 10 regionLink第45页，共62页，2022年，5月20日，9点58分，星期一The RNA polymerase core enyzme, a2bbw, has a general non-specific affinity for DNA, which is referred to as loose binding that is fairly stable.The addition of s factor to the core enzyme markedly reduces the holoenzyme af

39、finity for non-specific binding by 20 000-fold, and enhances the holoenzyme binding to correct promoter sites 100 times. Overall, s factor binding dramatically increases the specificity of the holoenzyme for correct promoter-binding site.The role of s factor in promoter binding B4 Transcription proc

40、ess第46页，共62页，2022年，5月20日，9点58分，星期一B4 Transcription process2. DNA 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 +1第47页，共62页，2022年，5月20日，9点58分，星期一3. RNA chain initiation+1The polymerase initially

41、 incorporates the first two nucleotides and forms a phosphodiester bond.B4 Transcription processStarts with a GTP or ATP第48页，共62页，2022年，5月20日，9点58分，星期一Abortive initiationThe RNA pol. goes through multiple abortive initiations before a successful initiation, which limits the overall rate of transcrip

42、tionThe minimum time for promoter clearance is 1-2 seconds (a long event, the synthesis is 40 nt/ sec)The first 9 nt are incorporated without polymerase movement along the DNA. Afterward, there is a significant probability that the chain will be aborted. B4 Transcription process第49页，共62页，2022年，5月20日

43、，9点58分，星期一4. RNA chain elongationB4 Transcription process第50页，共62页，2022年，5月20日，9点58分，星期一Promoter clearance: when initiation succeeds, the enzyme releases s factor and forms a ternary complex of polymerase-DNA-nascent RNA, causing the polymerase to progress along the DNA to allow the re-initiation of

44、 transcription.B4 Transcription process第51页，共62页，2022年，5月20日，9点58分，星期一Transcription bubble: containing 17 bp of unwound DNA region and the 3-end of the RNA that forms a hybrid helix about 12 bp.moves along the DNA with RNA polymerase which unwinds DNA at the front and rewinds it at the rear.The E. c

45、oli polymerase moves at an average rate of 40 nt per sec, depending on the local DNA sequence.B4 Transcription process第52页，共62页，2022年，5月20日，9点58分，星期一Transcription bubble第53页，共62页，2022年，5月20日，9点58分，星期一5. RNA chain termination Termination occurs at terminator DNA sequences. The most common stop signal

46、 is an RNA hairpin (self-complement structure) commonly GC-rich to favor the structure stability3. Rho-dependent and Rho-independent Termination. 第54页，共62页，2022年，5月20日，9点58分，星期一TerminatorA specific DNA sequence where the transcription complex dissociateRho protein (r) independent terminator contains: self-complementary region that is G-C rich and can form a stem-loop or hairpin secondary