Section K 原核轉(zhuǎn)錄

1、Section K: Transcription in prokaryotesK1 Basic principles of transcriptionK2 Escherichia coli RNA polymeraseK3 The E. coli s s70 promoterK4 transcription process. An overview, the process of RNA synthesis ( initiation, elongation, termination)Properties, a a subunit, b b subunit, b b subunit, sigma

2、 (s s) factorPromoter, s s70 size, -10 sequence, -35 sequence, transcription start site, promoter efficiencyPromoter binding, unwinding, RNA chain initiation, elongation, termination (r r factor)K1: Basic principles of transcription Transcription: an overview (comparison with replication) The proces

3、s of RNA synthesis: initiation, elongation, terminationK1-1: Transcription: an overviewKey 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 expression; for example, the coding region is d

4、ownstream of the initiation codon.Gene XPrimary transcriptm7GpppAAAAAn+1upstreamdownstreammRNAUpstream identifies sequences proceeding in the opposite direction from expression; for example, the bacterial promoter is upstream from the transcription unit, the initiation codon is upstream of the codin

5、g 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 RNA Terminator is a sequence of DNA, represented at the end of the tra

6、nscript, 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 transcript is the original unmodified RNA product corresponding to a transcription unit.Replication: synt

7、hesis 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 molecule using one of the two DNA strands as a template.1 DNA molecule 1 RNA moleculeReplication-synthesis of the leading strandth

8、e same direction as the replication fork movesReplication- Synthesis of the Okazaki fragmentsOpposite to the replication fork movementCoupling the synthesis of leading and lagging strands with a dimeric DNA pol III (E. coli)Transcription RNA synthesis occurs in the 53 direction and its sequence corr

9、esponds to the sense strand (coding strand). The template of RNA synthesis is the antisense strand (template strand). Phosphodiester bonds: same as in DNA Necessary components: RNA polymerase, transcription factors, rNTPs, promoter & terminator/templateK1-2: The process of RNA synthesis initiati

10、on elongation terminationFlowchart of RNA synthesisBack 1, 2PromoterTerminatorTranscribed regionSense strandAntisense strandDNARNATranscription+1Fig. 2. Structure of a typical transcription unitInitiation (template recognition) Binding of an RNA polymerase to the dsDNA Slide to find the promoter Unw

11、ind the DNA helix Synthesis of the RNA strand at the start site (initiation site), this position called position +1LinkElongation Covalently adds ribonucleotides to the 3-end of the growing RNA chain. The RNA polymerase extend the growing RNA chain in the direction of 5 3 The enzyme itself moves in

12、3 to 5 along the antisense DNA strand.LinkTermination Ending of RNA synthesis: the dissociation of the RNA polymerase and RNA chain from the template DNA at the terminator site. Terminator: often contains self-complementary regions which can form a stem-loop or hairpin structure in the RNA products

13、(see K4 for details)Terminator structureK2 Escherichia coli RNA polymerase E. coli RNA polymerase a a subunit b b subunit b b subunit sigma (s s) factorK2-1 E. coli RNA polymeraseSynthesis of single-stranded RNA from DNA template. Requires no primer for polymerization Requires DNA for activity and i

14、s most active with a double-stranded DNA as template. 5 3 synthesis Require Mg2+ for RNA synthesis activity lacks 3 5 exonuclease activity, and the error rate of nucleotides incorporation is 10-4 to 10-5. usually are multisubunit enzyme.RNA polymeraseE. coli polymerase E. coli has a single DNA-direc

15、ted RNA polymerase that synthesizes all types of RNA. One of the largest enzyme in the cells Consists of at least 5 subunits in the holoenzyme, 2 alpha (a a), and 1 of beta (b b), beta prime (b b), omega (w w) and sigma (s s) subunits Shaped as a cylindrical （圓柱形）（圓柱形）channel that can bind directly

16、to 16 bp of DNA. The whole polymerase binds over 60 bp. RNA synthesis rate: 40 nt per second at 37oCE. coli RNA polymeraseBoth initiation & elongationInitiation only36.5 KD36.5 KD151 KD155 KD11 KD70 KDThe polymerases of bacteriophage T3 and T7 are smaller single polypeptide chains, they synthesi

17、ze RNA rapidly (200 nt/sec) and recognize their own promoters which are different from E. coli promoters.RNA polymerase differs from organism to organism K2-2: a a subunitE. coli polymerase: a a subunit Two identical subunits in the core enzyme Encoded by the rpoA gene Required for assembly of the c

18、ore enzyme Plays a role in promoter recognition. plays a role in the interaction of RNA polymerase with some regulatory factorsK2-3&4: b b and b b subunitb b is encoded by rpoB gene, and b b is encoded by rpoC gene . Make up the catalytic center of the RNA polymeraseTheir sequences are related t

19、o those of the largest subunits of eukaryotic RNA polymerases, suggesting that there are common features to the actions of all RNA polymerases. mutations in rpoB affect all stages of transcription. Mutations in rpoC show that b b also is involved at all stages.b b subunit may contain two domains res

20、ponsible for transcription initiation and elongation Rifampicin (利福平利福平)：has been shown to bind to the subunit, and inhibit transcription initiation by prokaryotic RNA pol.1. Streptolydigins(利迪鏈菌素利迪鏈菌素)：resistant mutations are mapped to rpoB gene as well. Inhibits transcription elongation but not in

21、itiation. b b subunit Binds two Zn 2+ ions and may participate in the catalytic function of the polymerase Hyparin (肝素肝素)：binds to the b b subunit and inhibits transcription in vitro. Hyparin competes with DNA for binding to the polymerase.2. b b subunit may be responsible for binding to the templat

22、e DNA .K2-5: Sigma (s s) factor Many prokaryotes contain multiple s s factors to recognize different promoters. The most common s s factor in E. coli is s s70. Binding of the s s factor converts the core RNA pol into the holoenzyme. s s factor is critical in promoter recognition, by decreasing the a

23、ffinity of the core enzyme for non-specific DNA sites (104) and increasing the affinity for the corresponding promoter s s factor is released from the RNA pol after initiation (RNA chain is 8-9 nt) Less amount of s s factor is required in cells than that of the other subunits of the RNA pol. K3: The

24、 E. coli s s70 promoter Promoter s s70 size -10 sequence -35 sequence transcription start site promoter efficiencyK3-1: Promoter The specific short conserved DNA sequences: upstream from the transcribed sequence, and thus assigned a negative number (location) required for specific binding of RNA Pol

25、. and transcription initiation (function) Were first characterized through mutations that enhance or diminish the rate of transcription of gene Different promoters result in differing efficiencies of transcription initiation, which in turn regulate transcription. PromoterTerminatorTranscribed region

26、Sense strandAntisense strandDNARNATranscription+1K3-2,3&4: s 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 DNase Up to position 40: critical for promoter

27、function (mutagenesis analysis) -10 and 35 sequence: 6 bp each, particularly important for promoter function in E. coli-5-8 bp- GC T ATTGACATATAAT-16-18 bp-+1-35 sequence-10 sequence-10 sequence (Pribonow box)The most conserved sequence in s s70 promoters at which DNA unwinding is initiated by RNA P

28、ol.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 and 8 bp from position +1, and the

29、 distance is critical.-35 sequence: enhances recognition and interaction with the polymerase s s factor A conserved hexamer sequence 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

30、 in 90% of all promotersK3-5: Transcription start site Is a purine in 90% of all gene G is more common at position +1 than A There are usually a C and T on either side of the start nucleotide (i.e. CGT or CAT)The sequences of five E. coli promotersK3-6: promoter efficiencyThere is considerable varia

31、tion in sequence between different promoters, and the transcription efficiency can vary by up to 1000-fold . The 35 sequence constitutes a recognition region which enhances recognition and interaction with the polymerase s s factor. The -10 sequence is important for DNA unwinding. The sequence aroun

32、d the start site influence initiation efficiency. The sequence of the first 30 bases to be transcribed controls the rate at which the RNA polymerase clears the promoter, hence influences the rate of the transcription and the overall promoter strength.Some promoter sequence are not sufficiently simil

33、ar to the consensus sequence to be strongly transcribed under normal condition, thus activating factor is required for efficient initiation. Example: 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

34、enhance polymerase binding and transcription initiation.Weak promoters and activating factorK4 Transcription process Promoter binding DNA unwinding RNA chain initiation RNA chain elongation RNA chain termination (r r factor) Promoter bindingThe searching process is extremely rapidlyClosed complex: t

35、he initial complex of the polymerase with the base-paired promoter DNA)and 10 regionLink The RNA polymerase core enyzme, a a2 2bbbbw,w, has a general non-specific affinity for DNA, which is referred to as loose binding that is fairly stable. The addition of s s factor to the core enzyme markedly red

36、uces the holoenzyme affinity for non-specific binding by 20 000-fold, and enhances the holoenzyme binding to correct promoter sites 100 times. Overall, s s factor binding dramatically increases the specificity of the holoenzyme for correct promoter-binding site.The role of s s factor in promoter bin

37、ding 2. 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 It is necessary to unwind the DNA so that the antisense strand to become accessible for base pairing and RNA synthesis. Negative sup

38、ercoiling enhances the transcription of many genes, since it facilitates unwinding. Negative supercoiling & unwinding3. RNA chain initiation+1The polymerase initially incorporates the first two nucleotides and forms a phosphodiester bond.Starts with a GTP or ATPAbortive initiation The RNA pol. g

39、oes through multiple abortive initiations before a successful initiation, which limits the overall rate of transcription The 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. Afterw

40、ard, there is a significant probability that the chain will be aborted. 4. RNA chain elongation Promoter clearance: when initiation succeeds, the enzyme releases s s factor and forms a ternary complex（三重復(fù)（三重復(fù)合體）合體） of polymerase-DNA-nascent RNA, causing the polymerase to progress along the DNA to al

41、low the re-initiation of transcription.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.1. The E. coli polymerase moves at an average rate of 40 nt per sec, depending on the local DNA sequence.Transcription bubble5. RNA chain termination Termination occurs at terminator DNA sequences. The most common sto