分子生物學教學課件：Part 3 Chapter 12 Mechanisms of transcription 2

1、Topic 3:transcription in eukaryotesCHAPTER12: Mechanisms of TranscriptionMultiple Forms of Eukaryotic RNA PolymerasePromotersEnhancers and SilencersComparison of eukaryotic and prokaryotic RNA polymerasesEukaryotes: Three polymerase transcribes different class of genes: Pol I-large rRNA genes; Pol I

2、I-mRNA genes; Pol III- tRNA, 5S rRNA and small nuclear RNA genes (U6) Prokaryotes: one polymerase transcribes all genesMultiple Forms of Eukaryotic RNA PolymeraseRibosomal genes are different from other nuclear genesDifferent base composition from other nuclear genesUnusually repetitiveFound in diff

3、erent compartment, the nucleolusThere are at least two RNA polymerases operating in eukaryotic nucleiOne transcribes major ribosomal RNA genesOne or more to transcribe rest of nuclear genesSeparation of eukaryotic RNA polymerasesRoeder and Rutter subjected extracts from sea urchin embryos to DEAE-Se

4、phadex chromatography. Green, protein measured by A280; red, RNA polymerase activity measured by incorporation of labeled UMP into RNA; blue, ammonium sulfate concentration. (Source: Adapted from Nature 224:235, 1969.)Cellular localization of the three rat liver RNA polymerasesRoeder and Rutter subj

5、ected the polymerases found in the nucleoplasmic fraction (a) or nucleolar fraction (b) of rat liverto DEAE-Sephadex chromatography.(Source: Adapted from Proceedings of the National Academy of Sciences 65(3):67582,March 1970.)Separation of the Three Nuclear PolymerasesEukaryotic nuclei contain three

6、 RNA polymerasesThese can be separated by ion-exchange chromatographyRNA polymerase I found in nucleolusLocation suggests in transcribes rRNA genesRNA polymerases II and III are found in the nucleoplasmRoles of the Three RNA PolymerasesPolymerase I makes large rRNA precursorPolymerase II makes Heter

7、ogeneous nuclear RNA (hnRNA)Small nuclear RNAPolymerase III makes precursors to tRNAs, 5S rRNA and other small RNAa-amanitin鵝膏覃堿This work, by Roeder and colleagues in 1974, depended on a toxin called a-amanitin. This highly toxic substance is found in several poisonous mushrooms of the genus Amanita

8、（傘形毒菌素）, including A. phalloides（鬼筆鵝膏菌）, “the death cap,” and A. bisporigera（傘形毒菌）, which is called “the angel of death” because it is pure white and deadly poisonous. Both species have proven fatal to many inexperienced mushroom hunters.Sensitivity of purified RNA polymerase0.02ug/mL 20ug/mLWeinman

9、n and Roeder assayed RNA polymerases I (green), II (blue), and III (red) with increasing concentrations of a-amanitin. Polymerase II was 50% inhibited by about 0.02 mg/mL of the toxin, whereas polymerase III reached 50% inhibition only at about 20 mg/mL of toxin. Polymerase I retained full activity

10、even at an a-amanitin concentration of 200 mg/mL. (Source: Adapted From Proceedings of the National Academy of Sciences USA 71(5):17904, May 1974.)Effect of a-amanitin on small RNA synthesisRNA Polymerase Subunit StructuresPolymerase II StructureFor enzymes like eukaryotic RNA polymerases, can be di

11、fficult to tell: Which polypeptides copurify with polymerase activity Which are actually subunits of the enzymeTechnique to help determine whether a polypeptide copurifies or is a subunit is called epitope taggingEpitope TaggingAdd an extra domain to one subunitOther subunits normalPolymerase labele

12、d by growing in labeled amino acidsPurify with antibodyDenature with detergent and separate on a gelSubunit structure of yeast RNA polymerase IIPolymerase IIOriginal 10 subunits are placed in 3 groups:Core related in structure and function to bacterial core subunitsCommon found in all 3 nuclear RNA

13、polymerasesNonessential subunits conditionally dispensable for enzymatic activityCore SubunitsThree polypeptides, Rpb1, Rpb2, Rpb3 are absolutely required for enzyme activityThese are homologous to b-, b-, and a-subunitsBoth Rpb1 and b-subunit binds DNARpb2 and b-subunit are at or near the nucleotid

14、e-joining active siteRpb3 does not resemble a-subunitThere is one 20-amino acid subunit of great similarity2 subunits are about same size, same stoichiometry2 monomers per holoenzymeCommon SubunitsThere are five common subunitsRpb5Rpb6Rpb8Rpb10Rpb12Little known about functionThey are all found in al

15、l 3 polymerasesSuggests play roles fundamental in transcriptionSubunits Nonessential for ElongationRpb4 and Rpb7 Dissociate fairly easily from polymeraseFound in substoichiometric quantitiesMight shuttle from one polymerase II to anotherRpb4 may help anchor Rpb7 to the enzymeMutants without Rpb4 and

16、 Rpb7 transcribes well, but cannot initiate at a real promoterRpb7 is an essential subunit, so must not be completely absent in the mutantPromotersThree eukaryotic RNA polymerases have:Different structuresTranscribe different classes of genesExpect that the 3 polymerases would recognize different pr

17、omotersRNA polymerase II core promoters are made up of combinations of 4 different sequence elementsEukaryotic core promoter (40 nt): the minimal set of sequence elements required for accurate transcription initiation by the Pol II machinery in vitro The transcription in eukaryotesTFIIB recognition

18、element (BRE)The TATA element/boxInitiator (Inr)The downstream promoter element (DPE)Fig 12-12: Pol II core promoterCore Promoter ElementsIn addition to TATA box, core promoters are:TFIIB recognition element (BRE)Initiator (Inr)Downstream promoter element (DPE)At least one of the four core elements

19、is missing in most promotersTATA-less promoters tend to have DPEsPromoters for highly specialized genes tend to have TATA boxes Promoters for housekeeping genes tend to lack themTATA boxAbout 25-35 bp upstream of the start siteTATAAA-34 to -26+1mRNA start5Well-defined transcription startCore Promote

20、r Elements TATA BoxTATA box Found on the nontemplate strandVery similar to the prokaryotic -10 boxThere are frequently TATA-less promotersHousekeeping genes that are constitutively active in nearly all cells as they control common biochemical pathwaysDevelopmentally regulated genes InitiatorInstead

21、of a TATA box, some eukaryotic gene contain an alternative promoter element, called an initiator.Initiator is highly degenerative.5 Y Y A N T/A Y Y Y +1Y = pyrimidine (C or T) N = any Upstream ElementsUpstream promoter elements are usually found upstream of class II core promotersDiffer from core pr

22、omoters in binding to relatively gene-specific transcription factorsGC boxes bind transcription factor Sp1CCAAT boxes bind CTF (CCAAT-binding transcription factor)Upstream promoter elements can be orientation-independent, yet are relatively position-dependentCpG islandGenes coding for intermediary m

23、etabolism are transcribed at low rates, and do not contain a TATA box or initiator.Most genes of this type contain a CG-rich stretch of 20-50 nt within 100 bp upstream of the start site region.A transcription factor called SP1 recognizes these CG-rich region.Gives multiple alternative mRNA start sit

24、es.mRNAMultiple 5-start sitesCpG island100 bpDemethylation at the 5 end of the gene is necessary for transcription. CpG islands surround the promoters of constitutively expressed genes where they are unmethylated.CpG islands also are found at the promoters of some tissue-regulated genes.There are 29

25、,000 CpG islands in the human genome.Methylation of a CpG island prevents activation of a promoter within it.Repression is caused by proteins that bind to methylated CpG doublets. EnhancersLocated several Kb away from the start site. Usually 100-200 bp long, containing multiple 8- to 20-bp control e

26、lements.Cell-type specificDirection-less (invertible)+1EnhancerEnhancerStructure of the SV40 virus early control region.Effects of deletions in the immunoglobulin g2b H-chain enhancerMap of the cloned g2b gene. (b) Assay of expression of the g2b gene at the protein level. (c) Assay of transcription

27、of the g2b gene.Cell 33 (July 1983) p. 719)The enhancing element in the g2b gene is orientation and position-independent. Enhancers and SilencersThese are position- and orientation-independent DNA elements that stimulate or depress, respectively, transcription of associated genesAre often tissue-spe

28、cific in that they rely on tissue-specific DNA-binding proteins for their activitiesSome DNA elements can act either as enhancer or silencer depending on what is bound to itThe sequence elements other than the core promoter that are required to regulate the transcription efficiencyThose increasing t

29、ranscription:Promoter proximal elementsUpstream activator sequences (UASs)Enhancers（增強子）Those repressing elements: silencers （沉默子）boundary elements （邊界元件）insulators (絕緣體)Regulatory SequencesRNA Pol II forms a pre-initiation complex with GTFs at the promoterThe involved GTFIIs (general transcription

30、factor for Pol II)TFIID=TBP (TATA box binding protein) + TAFs (TBP association factors) TFIIA, B, F, E, H The transcription in eukaryotesTBP in TFIID binds to the TATA boxTFIIA and TFIIB are recruited with TFIIB binding to the BRERNA Pol II-TFIIF complex is then recruitedTFIIE and TFIIH then bind up

31、stream of Pol II to form the pre-initiation complex Promoter melting using energy from ATP hydrolysis by TFIIH )Promoter escapes after the phosphorylation of the CTD tailThe other GTFs also have specific roles in initiation 10 TAFs: (1) two of them bind DNA elements at the promoter (Inr and DPE); (2

32、) several are histone-like TAFs and might bind to DNA similar to that histone does; (3) one regulates the binding of TBP to DNA The transcription in eukaryotesTFIIB: (1) a single polypeptide chain, (2) asymmetric binding to TBP and the promoter DNA (BRE), (3)bridging TBP and the polymerase, (4) the

33、N-terminal inserting in the RNA exit channel resembles the s3.2 .TFIIF: (1) a two subunit factor, (2) binding of Pol II-TFIIF stabilizes the DNA-TBP-TFIIB complex, which is required for the followed factor bindingTFIIE: recruits and regulates TFIIHTFIIH: (1) controls the ATP-dependent transition of

34、the pre-initiation complex to the open complex, (2) contains 9 subunits and is the largest GTF; two functions as ATPase and one is protein kinase. (3) important for promoter melting and escape. (4) ATPase functions in nucleotide mismatch repair, called transcription-coupled repair. in vivo, transcri

35、ption initiation requires additional proteinsThe mediator complexTranscriptional regulatory proteinsNucleosome-modifying enzymesTo counter the real situation that the DNA template in vivo is packed into nucleosome and chromatin The transcription in eukaryotesassembly of the pre-initiation complex in

36、 presence of mediator, nucleosome modifiers and remodelers, and transcriptional activatorsMediator consists of many subunits, some conserved from yeast to humanMore than 20 subunits7 subunits show significant sequence homology between yeast and humanOnly subunit Srb4 is essential for transcription o

37、f essentially all Pol II genes in vivoOrganized in modules (模塊) The transcription in eukaryotescomparison of the yeast and human mediatorsEukaryotic RNA Pol II holoenzyme is a putative preformed complex:Pol II + mediator + some of GTFs Prokaryotic RNA Polymerase holoenzyme:core polymerase + s factor

38、A new set of factors stimulate Pol II elongation and RNA proofreading The transcription in eukaryotesThe 3 Ends of mRNAs Are Generated by Cleavage and PolyadenylationThe sequence AAUAAA is a signal for cleavage to generate a 3 end of mRNA that is polyadenylated.The reaction requires a protein complex that contains a specificity factor, an endonuclease, and poly(A) polymerase.The specificity factor and endonuclease cleave RNA downstream of AAUAAA.Function of poly(A) tailIncreased mRNA