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1、Topic 3:transcription in eukaryotesCHAPTER12: Mechanisms of TranscriptionlMultiple Forms of Eukaryotic RNA PolymeraselPromoterslEnhancers and SilencersComparison of eukaryotic and prokaryotic RNA polymerasesEukaryotes: Three polymerase transcribes different class of genes: Pol I-large rRNA genes; Po
2、l II-mRNA genes; Pol III- tRNA, 5S rRNA and small nuclear RNA genes (U6) Prokaryotes: one polymerase transcribes all genesMultiple Forms of Eukaryotic RNA PolymeraselRibosomal genes are different from other nuclear genesDifferent base composition from other nuclear genesUnusually repetitiveFound in
3、different compartment, the nucleoluslThere 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 DE
4、AE-Sephadex 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
5、 subjected 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 PolymeraseslEukaryotic nuclei contain
6、 three RNA polymerasesThese can be separated by ion-exchange chromatographylRNA polymerase I found in nucleolusLocation suggests in transcribes rRNA geneslRNA polymerases II and III are found in the nucleoplasmRoles of the Three RNA PolymeraseslPolymerase I makes large rRNA precursorlPolymerase II m
7、akes Heterogeneous nuclear RNA (hnRNA)Small nuclear RNAlPolymerase III makes precursors to tRNAs, 5S rRNA and other small RNAa a-amanitin鹅膏覃碱鹅膏覃碱This work, by Roeder and colleagues in 1974, depended on a toxin called a a-amanitin. This highly toxic substance is found in several poisonous mushrooms o
8、f the genus Amanita（伞形毒菌素）（伞形毒菌素）, including A. phalloides（鬼笔鹅（鬼笔鹅膏菌）膏菌）, “the death cap,” and A. bisporigera（伞形毒菌）（伞形毒菌）, 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
9、 RNA polymerase0.02ug/mL 20ug/mLWeinmann and Roeder assayed RNA polymerases I (green), II (blue), and III (red) with increasing concentrations of a a-amanitin. Polymerase II was 50% inhibited by about 0.02 m mg/mL of the toxin, whereas polymerase III reached 50% inhibition only at about 20 m mg/mL o
10、f toxin. Polymerase I retained full activity even at an a a-amanitin concentration of 200 m mg/mL. (Source: Adapted From Proceedings of the National Academy of Sciences USA 71(5):17904, May 1974.)Effect of a a-amanitin on small RNA synthesisRNA Polymerase Subunit StructuresPolymerase II StructurelFo
11、r enzymes like eukaryotic RNA polymerases, can be difficult to tell: Which polypeptides copurify with polymerase activity Which are actually subunits of the enzymelTechnique to help determine whether a polypeptide copurifies or is a subunit is called epitope taggingEpitope TagginglAdd an extra domai
12、n to one subunitlOther subunits normallPolymerase labeled by growing in labeled amino acidslPurify with antibodylDenature with detergent and separate on a gelSubunit structure of yeast RNA polymerase IIPolymerase IIOriginal 10 subunits are placed in 3 groups:lCore related in structure and function t
13、o bacterial core subunitslCommon found in all 3 nuclear RNA polymeraseslNonessential subunits conditionally dispensable for enzymatic activityCore SubunitslThree polypeptides, Rpb1, Rpb2, Rpb3 are absolutely required for enzyme activitylThese are homologous to b b-, b b-, and a a-subunitslBoth Rpb1
14、and b b-subunit binds DNAlRpb2 and b b-subunit are at or near the nucleotide-joining active sitelRpb3 does not resemble a a-subunitThere is one 20-amino acid subunit of great similarity2 subunits are about same size, same stoichiometry2 monomers per holoenzymeCommon SubunitslThere are five common su
15、bunitsRpb5Rpb6Rpb8Rpb10Rpb12lLittle known about functionlThey are all found in all 3 polymeraseslSuggests play roles fundamental in transcriptionSubunits Nonessential for ElongationlRpb4 and Rpb7 Dissociate fairly easily from polymeraseFound in substoichiometric quantitiesMight shuttle from one poly
16、merase II to anotherRpb4 may help anchor Rpb7 to the enzymeMutants without Rpb4 and Rpb7 transcribes well, but cannot initiate at a real promoterlRpb7 is an essential subunit, so must not be completely absent in the mutantPromoterslThree eukaryotic RNA polymerases have:Different structuresTranscribe
17、 different classes of geneslExpect that the 3 polymerases would recognize different promotersRNA 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 initiat
18、ion by the Pol II machinery in vitro The transcription in eukaryoteslTFIIB recognition element (BRE)lThe TATA element/boxlInitiator (Inr)lThe downstream promoter element (DPE)Fig 12-12: Pol II core promoterCore Promoter ElementslIn addition to TATA box, core promoters are:TFIIB recognition element (
19、BRE)Initiator (Inr)Downstream promoter element (DPE)lAt least one of the four core elements is missing in most promoterslTATA-less promoters tend to have DPEslPromoters for highly specialized genes tend to have TATA boxes lPromoters for housekeeping genes tend to lack themTATA boxAbout 25-35 bp upst
20、ream of the start siteTATAAA-34 to -26+1mRNA start5Well-defined transcription startCore Promoter Elements TATA BoxlTATA box Found on the nontemplate strandVery similar to the prokaryotic -10 boxThere are frequently TATA-less promoterslHousekeeping genes that are constitutively active in nearly all c
21、ells as they control common biochemical pathwayslDevelopmentally regulated genes InitiatorlInstead of a TATA box, some eukaryotic gene contain an alternative promoter element, called an initiator.lInitiator is highly degenerative.5 Y Y A N T/A Y Y Y +1Y = pyrimidine (C or T) N = any Upstream Element
22、slUpstream promoter elements are usually found upstream of class II core promoterslDiffer from core promoters in binding to relatively gene-specific transcription factorsGC boxes bind transcription factor Sp1CCAAT boxes bind CTF (CCAAT-binding transcription factor)lUpstream promoter elements can be
23、orientation-independent, yet are relatively position-dependentCpG islandlGenes coding for intermediary metabolism are transcribed at low rates, and do not contain a TATA box or initiator.lMost genes of this type contain a CG-rich stretch of 20-50 nt within 100 bp upstream of the start site region.lA
24、 transcription factor called SP1 recognizes these CG-rich region.lGives multiple alternative mRNA start sites.mRNAMultiple 5-start sitesCpG island100 bpDemethylation at the 5 end of the gene is necessary for transcription. lCpG islands surround the promoters of constitutively expressed genes where t
25、hey are unmethylated.lCpG islands also are found at the promoters of some tissue-regulated genes.lThere are 29,000 CpG islands in the human genome.lMethylation of a CpG island prevents activation of a promoter within it.lRepression is caused by proteins that bind to methylated CpG doublets. Enhancer
26、slLocated several Kb away from the start site. lUsually 100-200 bp long, containing multiple 8- to 20-bp control elements.lCell-type specificlDirection-less (invertible)+1EnhancerEnhancerStructure of the SV40 virus early control region.Effects of deletions in the immunoglobulin g g2b H-chain enhance
27、r(a)Map of the cloned g g2b gene. (b) Assay of expression of the g g2b gene at the protein level. (c) Assay of transcription of the g g2b gene.Cell 33 (July 1983) p. 719)The enhancing element in the g g2b gene is orientation and position-independent. Enhancers and SilencerslThese are position- and o
28、rientation-independent DNA elements that stimulate or depress, respectively, transcription of associated geneslAre often tissue-specific in that they rely on tissue-specific DNA-binding proteins for their activitieslSome DNA elements can act either as enhancer or silencer depending on what is bound
29、to itThe sequence elements other than the core promoter that are required to regulate the transcription efficiencyThose increasing transcription:lPromoter proximal elementslUpstream activator sequences (UASs)lEnhancers（增（增强强子）子）Those repressing elements: lsilencers （沉默子）（沉默子）lboundary elements （边界元件
30、）界元件）linsulators (绝缘体体)Regulatory SequencesRNA Pol II forms a pre-initiation complex with GTFs at the promoterThe involved GTFIIs (general transcription factor for Pol II)TFIID=TBP (TATA box binding protein) + TAFs (TBP association factors) TFIIA, B, F, E, H The transcription in eukaryotes1.TBP in T
31、FIID binds to the TATA box2.TFIIA and TFIIB are recruited with TFIIB binding to the BRE3.RNA Pol II-TFIIF complex is then recruited4.TFIIE and TFIIH then bind upstream of Pol II to form the pre-initiation complex 5.Promoter melting using energy from ATP hydrolysis by TFIIH )6.Promoter escapes after
32、the phosphorylation of the CTD tailThe other GTFs also have specific roles in initiationl 10 TAFs: (1) two of them bind DNA elements at the promoter (Inr and DPE); (2) 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 tr
33、anscription in eukaryotesnTFIIB: (1) a single polypeptide chain, (2) asymmetric binding to TBP and the promoter DNA (BRE), (3)bridging TBP and the polymerase, (4) the N-terminal inserting in the RNA exit channel resembles the s s3.2 .3.2 .lTFIIF: (1) a two subunit factor, (2) binding of Pol II-TFIIF
34、 stabilizes the DNA-TBP-TFIIB complex, which is required for the followed factor bindinglTFIIE: recruits and regulates TFIIHlTFIIH: (1) controls the ATP-dependent transition of the pre-initiation complex to the open complex, (2) contains 9 subunits and is the largest GTF; two functions as ATPase and
35、 one is protein kinase. (3) important for promoter melting and escape. (4) ATPase functions in nucleotide mismatch repair, called transcription-coupled repair. in vivo, transcription initiation requires additional proteinslThe mediator complexlTranscriptional regulatory proteinslNucleosome-modifying
36、 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 presence of mediator, nucleosome modifiers and remodelers, and transcriptional activatorsMediator consists of many subu
37、nits, some conserved from yeast to humanlMore than 20 subunitsl7 subunits show significant sequence homology between yeast and humanlOnly subunit Srb4 is essential for transcription of essentially all Pol II genes in vivolOrganized in modules (模块模块) The transcription in eukaryotescomparison of the y
38、east comparison of the yeast and human mediatorsand human mediatorsEukaryotic RNA Pol II holoenzyme is a putative preformed complex:Pol II + mediator + some of GTFs Prokaryotic RNA Polymerase holoenzyme:core polymerase + s s factorA new set of factors stimulate Pol II elongation and RNA proofreading
39、 The transcription in eukaryotesThe 3 Ends of mRNAs Are Generated by The 3 Ends of mRNAs Are Generated by Cleavage and PolyadenylationCleavage and PolyadenylationlThe sequence AAUAAA is a signal for cleavage to generate a 3 end of mRNA that is polyadenylated.lThe reaction requires a protein complex
40、that contains a specificity factor, an endonuclease, and poly(A) polymerase.lThe specificity factor and endonuclease cleave RNA downstream of AAUAAA.Function of poly(A) taillIncreased mRNA stabilitylIncreased translational efficiencylSplicing of last intronThe 5 End of Eukaryotic mRNA Is CappedlThe
41、5 cap of most mRNA is monomethylated, but some small noncoding RNAs are trimethylated.lThe cap structure is recognized by protein factors to influence mRNA stability, splicing, export, and translation.lA 5 cap is formed by adding a G to the terminal base of the transcript via a 55 link.lThe capping
42、process takes place during the transcription, which may be important for transcription reinitiation.Function of 5caplProtection from degradationlIncreased translational efficiencylTransport to cytoplasmlSplicing of first intronClass I PromoterslClass I promoters are not well conserved in sequence ac
43、ross specieslGeneral architecture of the promoter is well conserved two elements:Core element surrounding transcription start siteUpstream promoter element (UPE) 100 bp farther upstreamSpacing between these elements is importantClass III PromoterslRNA polymerase III transcribes a set of short geneslThese have promoters that lie wholly within the geneslThere are 3 types of these promotersPromoters of Some Polymerase III GeneslType I (5S rRNA) has 3 regions:Box AShort intermediate elementBox ClType II (tRNA) has 2 regions:Box A Box BlType III (nonclassical) resemble those of type II
