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1、Chapter 17Gene Regulation in Eukaryotes1. 1.Principles are the same: signals, activators and repressors, recruitment and allostery, cooperative binding2.2.Expression of a gene can be regulated at the similar steps, and the initiation of transcription is the most pervasively regulated step. Similarit
2、y of regulation between eukaryotes and prokaryote1. 1.Pre-mRNA splicing adds an important step for regulation.2.2.The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart.3.3.Nucleosomes and their modifiers influence access to genes.4.4.Many eukaryotic genes have mor
3、e regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes.Difference in regulation between eukaryotes and prokaryoteA lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration Fig. 17-1 The regulatory elements
4、 of a bacterial, yeast, and human gene.Enhancer:Enhancer: a given site binds regulator responsible a given site binds regulator responsible for activating the gene.for activating the gene.Alternative enhancerAlternative enhancer binds different groups of binds different groups of regulators and cont
5、rol expression of the same gene at regulators and control expression of the same gene at different times and places in responsible to different different times and places in responsible to different signals.signals.Activation at a distance is much more common in Activation at a distance is much more
6、 common in eukaryotes.eukaryotes. Insulators or boundary elements or boundary elements are regulatory are regulatory sequences to ensure a linked promoter not sequences to ensure a linked promoter not responding to the activator binding.responding to the activator binding. nMechanisms of Eukaryotic
7、RegulatorsnSignal Transduction and the Control of Transcriptional RegulatorsnGene “Silencing” by Modification of Histones and DNAnEukaryotic Gene Regulation at Steps after Transcription Initiation Structure of Eukaryotic Regulators Transcriptional Activation Transcriptional RepressorsStructure of Eu
8、karyotic Regulators Structure of Eukaryotic Regulators 1.The more elaborate transcriptional machinery 2.The nucleosome and their modifiers typical of eukaryotesActivators/RepressorsnSeparated DNA Binding and Activation FunctionsnDNA-Binding DomainsnActivating RegionsActivators Have Separate DNA Bind
9、ing and Activation Functionsn nEukaryotic activators have separate DNA binding and activating regions as well. The two surfaces are very often in separate domains of the protein. (Figure 17-2)FIGURE 17-2 Gal4 bound to its site on DNA.n nOne such gene is called GAL1. GAL4 binds to four sites located
10、275bp upstream of GAL1Gal4 is the most studied eukaryotic activatorGal4 activates transcription of the galactose genes in the yeast S. cerevisae.Gal4 binds to four sites upstream of GAL1, and activates transcription 1,000-fold in the presence of galactoseFig; 17-3 The regulatory sequences of the yea
11、st GAL1 gene.The separate DNA binding and activating domains of Gal4 were revealed in two complementary experiments1. 1. Expression of the Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein of the activator produces a protein bound to the DNA normally but di
12、d not activate bound to the DNA normally but did not activate transcription.transcription.2.2. Fusion of the Fusion of the C-terminal region (activation C-terminal region (activation domain)domain) of the activator to the DNA binding domain of the activator to the DNA binding domain of a bacterial r
13、epressor, of a bacterial repressor, LexALexA activates the transcription activates the transcription of the reporter gene. (of the reporter gene. (Domain swap experiment) Domain swap experiment Moving domains among proteins, proving that domains can be dissected into separate parts of the proteins.M
14、any similar experiments shows that DNA binding domains and activating regions are separable.Bactrial regulatory proteins1.Most use the helix-turn-helix motif to bind DNA target2.Most bind as dimers to DNA sequence: each monomer inserts an a helix into the major groove. Eukaryotic regulatory proteins
15、1.Recognize the DNA using the similar principles, with some variations in detail.2.Some form heterodimers to recognize DNA, extending the range of DNA-binding specificity. DNA binding domainsn nHomeodomain proteinsn nZinc containing DNA-binding domain: zinc finger and zinc clustern nLeucine zipper m
16、otifn nHelix-Loop-Helix proteins : basic zipper and HLH proteinsHomeodomain proteins: The homeodomain is a class of helix-turn-helix DNA-binding The homeodomain is a class of helix-turn-helix DNA-binding domain and recognizes DNA in essentially the same way as domain and recognizes DNA in essentiall
17、y the same way as those bacterial proteinsthose bacterial proteinsZinc containing DNA-binding domains finger domain: Zinc finger proteins (TFIIIA) Zinc cluster domain (Gal4)Leucine Zipper Motif: The Motif combines dimerization and DNA-binding surfaces within a single structural unit.Dimerization is
18、mediated by hydrophobic Dimerization is mediated by hydrophobic interactions between the appropriately-spaced interactions between the appropriately-spaced leucine to form a leucine to form a coiled coilcoiled coil structure structureHelix-Loop-Helix motif:Helix-loop-helix proteins. An extended heli
19、cal region from each of two monomers insets into the major groove of the DNA. Because the region of the a-helix that binds DNA contains baisc amino acids residues, Leucine zipper and HLH proteins are often called basic zipper and basic HLH proteins.Both of these proteins use hydrophobic amino acid r
20、esidues for dimerization.Activating regionsThe activating regions are grouped on the basis of amino acids contentAcidic activation domains Glutamine-rich domains Proline-rich domainsnMechanisms of Eukaryotic Regulators Structure of Eukaryotic Regulators Mechanisms of Eukaryotic Activator Mechanisms
21、of Eukaryotic repressornSignal Transduction and the Control of Transcriptional RegulatorsnGene “Silencing” by Modification of Histones and DNAnEukaryotic Gene Regulation at Steps after Transcription InitiationMechanisms of Eukaryotic Activator Eukaryotic activators also work by recruiting as in bact
22、eria, but recruit polymerase indirectly in two ways : 1. Interacting with parts of the transcription Interacting with parts of the transcription machinery.machinery. 2. Recruiting nucleosome modifiers that alter 2. Recruiting nucleosome modifiers that alter chromatin in the vicinity of a gene.chroma
23、tin in the vicinity of a gene. 1. Recruitment of the transcription machinery.Recruitment of the transcription machinery. The eukaryotic The eukaryotic transcriptional machinerytranscriptional machinery contains polymerase contains polymerase and numerous proteins being organized to several complexes
24、, and numerous proteins being organized to several complexes, such as the such as the MediatorMediator and the and the TFTFD complexD complex. . ActivatorsActivators interact with one or more of these complexes and recruit them interact with one or more of these complexes and recruit them to the gen
25、e.to the gene.1. 1.TBP in TFIIDTBP in TFIID binds to the TATA binds to the TATA boxbox2. 2.TFIIA and TFIIBTFIIA and TFIIB are recruited are recruited with TFIIB binding to the BREwith TFIIB binding to the BRE3. 3.RNA Pol II-TFIIFRNA Pol II-TFIIF complex is then complex is then recruitedrecruited4. 4
26、.TFIIE and TFIIHTFIIE and TFIIH then bind then bind upstreamupstream of Pol II to form the pre- of Pol II to form the pre-initiation complex initiation complex 5. 5.Promoter meltingPromoter melting using energy using energy from ATP hydrolysis by TFIIH )from ATP hydrolysis by TFIIH )6. 6.Promoter es
27、capesPromoter escapes after the after the phosphorylation of the CTD tailphosphorylation of the CTD tailActivator Bypass Experiment-Activator Bypass Experiment-Activation of Activation of transcription through direct tethering of transcription through direct tethering of mediator to DNA.mediator to
28、DNA. Directly fuse the bacterial DNA-binding protein LexA protein to the mediator complex Gal11 to activate GAL1 expression. 2. Activators also recruit Nuleosome modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome modifiers :1. Those add chemical groups to the
29、 tails of histones, such as histone acetyl transferases (HATs)2. Those remodel the nucleosomes, such as the ATP-dependent activity of SWI5/SNFLocal alterations in chromatin directed by activatorsLocal alterations in chromatin directed by activatorsModification of the N-terminal tails of the histones
30、Modification of the histone N-terminal tails alters the function of chromatinFig 7-35 Nucleosome movement catalyzed by nucleosome remodeling complexesremodling Effect of histone tail modification Many enkaryotic activatorsparticularly in higher eukaryoteswork from a distance.1. 1.Some proteins help,
31、 for example Chip protein in Some proteins help, for example Chip protein in DrosophilaDrosophila. . 2.2.The compacted chromosome structure help. DNA The compacted chromosome structure help. DNA is wrappedis wrapped in in nucleosomesnucleosomes in eukaryotes.So sites in eukaryotes.So sites separated
32、 by many base pairs may not be as far separated by many base pairs may not be as far apart in the cell as thought.apart in the cell as thought.3. Action at a distance: loops and insulatorsInsulators block activation by enhancersSpecific elements called insulators control the actions of activators, p
33、reventing the activating the non-specific genes Transcriptional SilencingnSilencing is a specializes form of repression that can spread along chromatin, switching off multiple genes without the need for each to bear binding sites for specific repressor.nInsulator elements can block this spreading, s
34、o insulators protect genes from both indiscriminate activation and repression. 4 Appropriate regulation of some groups of genes requires locus control region (LCR). A group of regulatory elements collectively called the locus control region (LCR), is found 30-50 kb upstream of the cluster of globin
35、genes. Its made up of multiple-sequence elements : something like enhancers, insulators or promoters. It binds regulatory proteins that cause the chromatin structure to “open up”, allowing access to the array of regulators.nMechanisms of Eukaryotic Regulators Structure of Eukaryotic Regulators Mecha
36、nisms of Eukaryotic Activator Mechanisms of Eukaryotic repressornSignal Transduction and the Control of Transcriptional RegulatorsnGene “Silencing” by Modification of Histones and DNAnEukaryotic Gene Regulation at Steps after Transcription Initiation Signal Integration and Combinatorial Control1. in
37、tegrate signalsnIn eukaryotic cells, numerous signals are often required to switch a gene on. So at many genes multiple activators must work together.nThey do these by working synergistically: two activators working together is greater than the sum of each of them working alone.Three strategies of s
38、ynergy :1.Two activators recruit a single complex 2.Activators help each other binding cooperativity 3.One activator recruit something that helps the second activator binda.“Classical” cooperative bindingb. Both proteins interacting with a third proteinc. A protein recruits a remodeller to reveal a
39、binding site for another proteind. Binding a protein unwinds the DNA from nucleosome a little, revealing the binding site for another proteinnSignal integration: the HO gene is controlled by two regulators; one recruits nucleosome modifiers and the other recruits mediatornSignal integration: Coopera
40、tive binding of activators at the human b-interferon gene.The examples of integrate signalsThe The HOHO gene gene is involved in the budding of yeast. is involved in the budding of yeast.It has two It has two activatorsactivators : : SWI5SWI5 and and SBFSBF. .alter the nucleosomeActive only at corre
41、ct stage of cell cycle The human -interferon gene is activated in cells upon The human -interferon gene is activated in cells upon viral infection. Infection triggers three activators :viral infection. Infection triggers three activators :NFB, IRF, and Jun/ATFThey bind cooperatively to sites within
42、an enhancer, form a structure called Enhanceosome.nCombinatory control lies at the heart of the complexity and diversity of eukaryotesnCombinatory control of the mating-type genes from S. cerevisiae2.Combinatory control There is extensive combinatorial control in eukaryotes.In complex multicellular
43、organisms, combinatorial control involves many more regulators and genes than shown above, and repressors as well as activators can be involved. Four signalsThree signalsnThe yeast S.cerevisiae exists in three forms: two haploid cells of different mating types a and a and the diploid formed when an
44、a and an a cell mate and fuse.nCells of the two mating types differ because they express different sets of genes : a specific genes and a specific genes.Combinatory control of the mating-type genes from S. cerevisiaena cell make the regulatory protein a1,a cell make the protein a1 and a2. A fourth r
45、egulator protein Mcm1 is also involved in regulatory the mating-type specific genes and is present in both cell types.Control of cell-type specific genes in yeastnIn eukaryotes, repressors dont work by binding to sites that overlap the promoter and thus block binding of polymerase, but most common w
46、ork by recruiting nucleosome modifiers.nFor example, histone deacetylases repress transcription by removing actetyl groups from the tails of histone.Transcriptional RepressorsWays in which eukaryotic repressor Work a and bWays in which eukaryotic repressor Work c and dFigure 17-19nIn the presence of
47、 glucose, Mig1 binds a site between the USAG and the GAL1 promoter. By recruiting the Tup1 repressing complex, Mig1 represses expression of GAL1. nTwo mechanisms have been proposed to explain the repressing effect of Tup1. First, Tup1 recruits histone deaxetylases. Second, Tup1 interacts directly wi
48、th the transcription machinery at the promoter and inhibits initiation.A specific example: Repression of the GAL1 gene in yeastnMechanisms of Eukaryotic RegulatorsnSignal Transduction and the Control of Transcriptional RegulatorsnGene “Silencing” by Modification of Histones and DNAnEukaryotic Gene R
49、egulation at Steps after Transcription Initiation Structure of Eukaryotic Regulators Transcriptional Activation Transcriptional RepressorsSignal Transduction and the Control of Transcriptional Regulators1 .Signals are often communicated to transcriptional regulators through signal transduction pathw
50、ay2 .Signals control the activities of eukaryotic transcriptional regulators in a variety of ways1.signal transduction involve STATa. The STAT pathwaya. The STAT pathwayThe JAK/STAT Signaling Pathway 2. The MAPK signalling pathways The RAS-activated MAPK pathway: RAS-RAF-MEK-MAPKrepresents the first
51、 example where all the steps in a complete signalling cascade from the cell surface receptor PTK(protein tyrosine kinase ), to the nuclear transcription is known.Signals control the activities of eukaryotic transcriptional regulators in a variety of waysOnce a signal has been communicated, directly
52、or indirectly, to a transcriptional regulator, how does it control the activity of that regulator ?In eukaryotes, transcriptional regulators are not typically controlled at the level of DNA binding. They are usually controlled in one of two basic ways : nUnmasking an activating regionn Transport in
53、or out of the nucleusActivator Gal4 is regulated by masking protein Gal80The signalling ligand causes activators (or repressors) to move to the nucleus where they act from cytoplasm.nMechanisms of Eukaryotic RegulatorsnSignal Transduction and the Control of Transcriptional RegulatorsnGene “Silencing
54、” by Modification of Histones and DNAnEukaryotic Gene Regulation at Steps after Transcription InitiationGene “silencing” is a position effecta gene is silenced because of where it is located, not in response to a specific environmental signal.The most common form of silencing is associated with a de
55、nse form of chromatin called heterochromatin. It is frequently associated with particular regions of the chromosome, notably the telomeres, and the centromeres.nGene “Silencing” by Modification of Histones and DNAThe The telomerestelomeres, the , the silent mating-type locussilent mating-type locus,
56、 and the , and the rDNA genesrDNA genes are all are all “silent”“silent” regions in regions in S.cerevisiaeS.cerevisiae. .Three genes encoding regulators of silencing, Three genes encoding regulators of silencing, SIR2SIR2, , 3 3, and , and 4 4 have been found ( have been found (SIRSIR stand for sta
57、nd for s silent ilent i information nformation r regulator).egulator).Silencing at the yeast telomere A histone code D Different patterns of modification on ifferent patterns of modification on histonehistone tails tails can be can be “read”“read” to mean different things. The to mean different thin
58、gs. The “meaning”“meaning” would be the result of the direct effects would be the result of the direct effects of these modifications on of these modifications on chromatin density and chromatin density and formform. . But in addition, the particular pattern of But in addition, the particular patter
59、n of modifications at any given location would recruit modifications at any given location would recruit specific proteins.specific proteins.Modification of the histone N-terminal tails alters the function of chromatinHistone modification and the histone code hypothesisn nTranscription can also be s
60、ilenced by Transcription can also be silenced by methylationmethylation of DNA by enzymes called of DNA by enzymes called DNA DNA methylasesmethylases. .n nThis kind of silencing is not found in yeast but This kind of silencing is not found in yeast but is common in mammalian cells.is common in mamm
61、alian cells.n nMethylationMethylation of DNA sequence can of DNA sequence can inhibit inhibit binding of proteinsbinding of proteins, including the , including the transcriptional machinery, and thereby block transcriptional machinery, and thereby block gene expression.gene expression.DNA methylases
62、.Switching a gene off :A mammalian gene marked by A mammalian gene marked by methylationmethylation of nearby DNA sequence of nearby DNA sequence recognized by recognized by DNA-binding proteinsDNA-binding proteinsrecruit recruit histonehistone deacetylasesdeacetylases and and histonehistone methyla
63、sesmethylasesmodify nearby chromatinmodify nearby chromatinThis gene is completely off.Switching a gene offPatterns of gene expression must sometimes be Patterns of gene expression must sometimes be inheritedinherited. These may remain for many cell . These may remain for many cell generationsgenera
64、tions, even if the signal that induced them is , even if the signal that induced them is present only present only fleetinglyfleetingly. . This inheritance of gene expression patterns is This inheritance of gene expression patterns is called called epigenetic regulationepigenetic regulation. . .epig
65、enetic regulationNucleosome and DNA modifications can provide the basis for epigenetic inheritance.DNA methylation is even more reliably inherited, but far more efficiently is the so-called maintenance methylases modify hemimethylated DNAthe very substrate provided by replication of fully methylated
66、 DNA. Patterns of DNA methylation can be maintained through cell divisionnMechanisms of Eukaryotic RegulatorsnSignal Transduction and the Control of Transcriptional RegulatorsnGene “Silencing” by Modification of Histones and DNAnEukaryotic Gene Regulation at Steps after Transcription InitiationnAfte
67、r transcription initiationnAfter transcriptionnTranslationn nIn eukaryotic cells, some regulational proteins aim at elongation.n nAt some genes there are sequence downstream of the promoter that cause pausing or stalling of the polymerase soon after initiation.n nAt those genes, the presence or abse
68、nce of certain elongation factors greatly influences the level at which the gene is expressed.nAfter transcriptionAlternative splicing:Alternative splicing:Early transcriptional regulation of Sxl in male and female fliesnMechanisms of Eukaryotic RegulatorsnSignal Transduction and the Control of Transcriptional RegulatorsnGene “Silencing” by Modification of Histones and DNAnEukaryotic Gene Regulation at Steps after Transcription Initiation
