分子生物学6真核生物的转录及其调控课件.ppt

1、Transcription in EukaryotesThe three RNA Polymerasescharacterization and functionMain Features of eukaryotic transcription1.The mechanism of eukaryotic transcription is similar to that in prokaryotes.2.A lot more proteins are associated with the eukaryotic transcription machinery,which results in th

2、e much more complicated transcription.3.Three eukaryotic polymerases transcribe different sets of genes.The activities of these polymerases are distinguished by their sensitivities to the fungal toxin-amanitin.4.In addition,eukaryotic cells contain additional RNA Pols in mitochondria and chloroplast

3、s.Three eukaryotic polymerasesType Location Substrate -amanitinRNA Pol INucleoliMost rRNAs geneInsensitiveRNA Pol IINucleoplasmAll protein-codinggenes and somesnRNA genesVerysensitiveRNA Pol IIINucleoplasmtRNAs,5S rRNA,U6 snRNA andother small RNAsModeratelysensitive-amanitinThe sensitivities of thre

4、e kinds of RNA polymerases on-amanitinRNA polymerase subunitsEach eukaryotic polymerase contains 12 or more subunits.the two largest subunits are similar to each other and to the and subunits of E.coli RNA Pol.There is one other subunit in all three RNA Pol homologous to subunit of E.coli RNA Pol.Fi

5、ve additional subunits are common to all three polymerases.Each RNA Pol contains additional four or seven specific subunit.RNA polymerase activities1.Transcription mechanism is similar to that of E.coli polymerase.Direction:5 32.Different from bacterial polymerasae,they require accessory factors for

6、 DNA bindThe CTD of RNA pol II1.The C-terminus of RNA Pol II contains a stretch of seven amino acids that is repeated 52 times in mouse enzyme and 26 times in yeast.2.The heptapeptide sequenc is:Tyr-Ser-Pro-Thr-Ser-Pro-Ser3.This repeated sequence is known as carboxyl terminal domain(CTD)4.The CTD se

7、quence may be phosphorylated at the serines and some tyrosines5.The CTD is unphosphorylated at transcription initiation,and phosphorylation occurs during transcription elongation as the RNA Pol II leaves the promoter(In vitro results).6.Because it transcribes all eukaryotic protein-coding gene,RNA P

8、ol II is the most important RNA polymerase for the study of differential gene expression.The CTD is an important target for differential activation of transcription elongation.RNA Pol I genes:the ribosomal repeatsRibosomal RNA Genes&nucleolus1.A copy of 18S,5.8S and 28S rRNA genes is organized as a

9、single transcription unit in eukayotes.A 45S rRNA transcript(13 000 nt long)is produced during transcription,which is then processed into 18S,5.8S and 28S rRNA.2.Pre-rRNA transcription units are arranged in clusters in the genome as long tandem arrays separated by nontranscribed spacer squences.3.Co

10、ntinuous transcription of multiple copies of rRNA genes by RNA Pol I is essential to produce sufficient rRNAs which are packaged into ribosomes.4.The arrays of rRNA genes(rRNA cluster)loop together to form the nucleolus and are known as nucleolar organizer regions.5.During active rRNA synthesis,the

11、pre-rRNA transcripts are packaged along the rRNA genes,visualizing in the electronic microscope as“Christmas tree structures”.RNA Pol I promoters1.Generally consists of a bipartite sequence in the region preceding the start site,including core element and the upstream control elements(UCE).2.RNA Pol

12、 I promoters in human cells are best characterized.Core element:-45 to+20,sufficient for transcription initiatiation.UCE:-180 to-107,to increase the transcription efficiency.Both regions are rich in G:C,with 85%identity.Upstream binding factor(UBF)A specific DNA-binding protein that binds to UCE,as

13、well as a different site in the upstream of the core element,causing the DNA to loop between the two sites.(two binding sites have no obvious similarity)UBF is essential for high level of transcription,and low level of expression occurs in its absence.1.Does not bind to promoters by itself2.Binds to

14、 and stabilizes the UBF-DNA complex.3.Interacts with the free downstream part of the core element.4.Recruit RNA Pol I to bind and to initiate the transcription.Selectivity factor 1(SL1)SL1 consists of 4 proteins1.TBP(TATA-binding protein)a factor also required for initiation by RNA Pol II and III.A

15、critical general factor in eukaryotic transcription that ensures RNA Pol to be properly localized at the startpoint.2.Other three subunits are referred to as TBP-associated factors(TAFIs)that are specific for RNA Pol I transcription.Other rRNA genesIn a simple eukaryote,Acanthamoeba,the rRNA genes h

16、ave only one control element(promoter)around 12-72 bp upstream from the transcription start site.Simple initiationTIF-1(homolog of SL-1)binds to the promoter RNA Pol I bind TIF-1 remains bound and the RNA Pol I is released for elongation.RNA Pol III genes:5S and tRNA transcriptionRNA Pol III1.Contai

17、ns at least 16 or more subunits2.Is located in nucleoplasm3.Synthesizes the precursors of 5S rRNA,the tRNAs and other small nuclear and cytosolic RNAsPromoters for RNA polymerase IIIMay consist of bipartite sequences downstream of the startpoint,with boxA separated from either boxC or boxB.Or they m

18、ay consist of separated sequences upstream of the startpoint(Oct,PSE,TATA).tRNA genes1.The initial transcripts of tRNA genes need to be processed to produce the mature tRNA.2.The transcription control regions of tRNA lies after the start site within the transcribed region.The two highly conserved co

19、ntrol sequences are called A box (5-TGGCNNAGTGG)B box (5-GGTTCGANNCC)A box and B box also encode important sequences in the tRNA itself,the D-loop and TC-loop.Therefore,the highly conserved sequence in tRNAs are also highly conserved promoter DNA sequences.3.Two complex DNA-binding factors required

20、for tRNA transcription initiationTF III C-binds to both the A and B boxes,an assembly factor for positioning TF III B.TFIII B(1)binds 50 bp upstream from the A box,but has no sequence specificity and the binding position is determined by the DNA bound TFIIIC.(2)consists of three subunits,one of whic

21、h is TBP,the general initiation factor;the second is called BRF(TFIIB-related factor);and the third is called B”.5S rRNA genes1.Tandemly arranged in a gene cluster.(In human,there is a single cluster of around 2000 genes.)2.Transcription control regions(promoters)are organized similar to those of tR

22、NA,except that C box is in place of B box.C box:+81-99 bp;A box:+50-653.Transcription factors(1)The C box acts as the binding site for TF III A.(2)TFIIIA acts as an assembly factor which allows TF III C to interact with the 5S rRNA promoter.(3)The A box may also stabilize TFIIIC binding.(4)TFIIIC is

23、 then bound to DNA site near+1.(5)TFIIIB and TFIIIC interact to recruit RNA Pol III to initiate transcription.Alternative RNA Pol III promotersMany RNA Pol III genes also rely onupstream sequences for regulation oftheir transcriptione.g.U6 snRNA and Epstein-Barr virusUse only regulatory genes upstre

24、am from their transcription start sites.U6 snRNA1.The coding region contains a characteristic A box that is not required for transcription.2.The upstream sequence contains sequences typical of RNA Pol II promoters,including a TATA box at bases 30 to 23.3.Shares several other transcription factor bin

25、ding sequences with many U RNA genes which are transcribed by RNA Pol IISuggestion:common transcription factors can regulate both RNA Pol II and Pol III genesRNA Pol III terminationThe RNA polymerase can terminate transcription without accessory factors.A cluster of A residue is often sufficient for

26、 termination.Xenopus borealis terminator:5-GCAAAAGC-3RNA Pol II genes:promoters and enhancersRNA Pol II1.located in nucleoplasm2.catalyzing the synthesis of the mRNA precursors for all protein-coding genes.3.RNA Pol-transcribed pre-mRNAs are processed through cap addition,poly(A)tail addition and sp

27、licing.PromotersMost promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription.It has a 7 bp consensus sequence 5-TATA(A/T)A(A/T)-3TBP binds to TATA box that includes an additional downstream bp(8 bp).-TATA box acts in a similar way to an E.coli p

28、romoter 10 sequence to position the RNA Pol II for correct transcription initiation.-The spacing but not the sequence between the TATA box and the start site is important.-Transcription starts with an adenine 50%of the time.-Some eukaryotic genes contain an initiator element instead of a TATA box.Th

29、e initiator element is located around the transcription start site.-Other genes have neither a TATA box nor an initiator element,and usually are transcribed at very low rates.Upstream regulatory elementsThe basal elements(the TATA box and initiator elements):primarily determine the location of the s

30、tartpoint,and sponsor initiation only at a rather low level.Upstream regulatory elements(URE)such as the SP1 box and CCAAT boxes,greatly increase the frequency of initiation.URE is located within 100-200 bp from the promoter,and plays an important role in ensuring efficient transcription.EnhancersSe

31、quence elements which can activate transcription from thousands of base pairs upstream or downstream.General characteristics of Enhancers1.Exert strong activation of transcription of a linked gene from the correct start site.2.activate transcription when placed in either orientation with respect to

32、linked genes3.Able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start site.4.Exert preferential stimulation of the closest of two tandem promotersGeneral transcription factorsand RNA Pol initiationTF II DMultiprotein Complex,includ

33、ing TBP,other proteins are known as TAFIIs.TBP is the only protein binds to TATA boxTBP1.a general transcription factor bound to DNA at the TATA box.2.a general transcription required by all 3 RNA pol.3.has a saddle structure with an overall dyad symmetry.TF II Abinds to TF II Dstabilizes TF II D-DN

34、A complexcontains at least 3 subunitsTF II B&RNA Pol bindingbinds to TF II DBinds to RNA Pol with TF II FTF II E,TF II J,TF II H binding Necessary for transcriptionphosphorylation of the polymerase CTD by TF II HFormation of a processive RNA polymerasecomplex and allows the RNA Pol to leave thepromo

35、ter region.The initiator transcription complexFor TATA-box lacking RNA Pol II promoters,TBP is recruited to the initiator element 0verlapping the start site by some DNA-binding proteins,TBP then recruit the other transcription factors and polymerase similar to TATA box gene transcription.Transcripti

36、on of a single gene may be regulated by many different factors interacting with regulatory elements upstream or downstream of the transcribed sequence.Gene XStart site+1Regulatory elements to bind transcription factorsEukaryotic Transcription Factors Common features of transcription factors1.bind sp

37、ecifically to some DNA sites:specific motifs in promoters,upstream regulatory elements(UREs)or enhancer regions.Some factors modulate transcription by protein-protein interaction 2.Activate/repress transcription.The activity of a transcription factor can be assigned to separate protein domainsnactiv

38、ation domains.nDNA-binding domains.ndimerization domains.Many transcription factors occur as homo-or heterodimers,held together by dimerization domains.nligand-binding domains.Allowing regulation of transcription factor activity by binding of an accessory small molecule.The steroid hormone receptors

39、 are an example containing all four of these types of domain.Domain swap experiments moving domains among proteins,proving that domains can be dissected into separate parts of the proteins.The experiment of fusing activation domains of yeast transcription factors Gal4 and Gcn4 into the bacterial Lex

40、A repressorTranscription activation domains are separable from their DNA binding activity.DNA-binding domainsHelix-turn-helix domainExamples of helix-turn-helix domains1.Homeodomain:encoded by a sequence called the homeobox,containing a 60-amino-acid.In the Antennapedia transcription factor of Droso

41、phila,this domain consists of four-helices in which helices and are at right angles to each other and are separated by a characteristic-turn.2.Other DNA-binding proteins such as the phage cro repressor,lac and trp repressors,and cAMP receptor protein,CRP.nThe recognition helix of the domain structur

42、e lies partly in the major groove and interacts with the DNA.nThe recognition helices of two homeodomain factors Bicoid and Antennapedia can be exchanged,and this swaps their DNA-binding specificities.The zinc finger domainZinc finger domain exists in two formsC2H2 zinc finger:a loop of 12 amino aci

43、ds anchored by two cysteine and two histidine residues that tetrahedrally co-ordinate a zinc ion.This motif folds into a compact structure comprising two-strands and one-helix.The-helix containing conserved basic amino acids binds in the major groove of DNA Examples(1)TFIIIA,the RNA Pol III transcri

44、ption factor,with C2H2 zinc finger repeated 9 times.(2)SP1,with 3 copies of C2H2 zinc finger.Usually,three or more C2H2 zinc fingers are required for DNA binding.C4 zinc finger:zinc ion is coordinated by 4 cysteine residues.Example steriod hormone receptor transcription factors,consisting of homo-or

45、 hetero-dimers,in which each monomer contains two C4 zinc finger.The basic domainnRich in basic amino acid residuesnfound in a number of DNA-binding proteins ngenerally associated with one or other of two dimerization domains,the leucine zipper or the helix-loop-helix(HLH)motif,resulting in basic le

46、ucine zipper(bZIP)or basic HLH proteins.Dimerization of the proteins brings together two basic domains which can then interact with DNA.Dimerization domainsLeucine zippernLeucine zipper proteins contain a hydrophobic leucine residue at every seventh position in a region that is often at the C-termin

47、al part of the DNA-binding domain.nThese leucines are responsible for dimerization through interaction between the hydrophobic faces of the-helices.This interaction forms a coiled-coil structurebZIP(basic leucine zipper)transcription factors:contain a basic DNA-binding domain N-terminal to the leuci

48、ne zipper.The N-terminal basic domains of each helix form a symmetrical structure in which each basic domains lies along the DNA in opposite direction,interacting with a symmetrical DNA recognition site with the zippered protein clampnThe leucine zipper is also used as a dimerization domain in prote

49、ins containing DNA-binding domains other than the basic domain,including some homeodomain proteins.The helix-loop-helix domain(HLH)nThe overall structure is similar to the leucine zipper,except that a nonhelical loop of polypeptide chain separates two-helices in each monomeric protein.nHydrophobic r

50、esidues on one side of the C-terminal-helix allow dimerization.nExample MyoD family of proteins.nSimilar to leucine zipper,the HLH motif is often found adjacent to a basic domain that requires dimerization for DNA binding.nBasic HLH proteins and bZIP proteins can form heterodimers allowing much grea