分子生物學張海紅chapter17

1、Chapter 17 Gene Regulation in Eukaryotes,Principles are the same: signals, activators and repressors, recruitment and allostery, cooperative binding Expression of a gene can be regulated at the similar steps, and the initiation of transcription is the most pervasively regulated step.,Similarity of r

2、egulation between eukaryotes and prokaryote,Pre-mRNA splicing adds an important step for regulation. The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart. Nucleosomes and their modifiers influence access to genes. Many eukaryotic genes have more regulatory bindin

3、g sites and are controlled by more regulatory proteins than are bacterial genes.,Difference in regulation between eukaryotes and prokaryote,A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration,Fig. 17-1 The regulatory elements of a bacterial,

4、yeast, and human gene.,Enhancer: a given site binds regulator responsible for activating the gene. Alternative enhancer binds different groups of regulators and control expression of the same gene at different times and places in responsible to different signals. Activation at a distance is much mor

5、e common in eukaryotes. Insulators or boundary elements are regulatory sequences to ensure a linked promoter not responding to the activator binding.,Mechanisms of Eukaryotic Regulators Signal Transduction and the Control of Transcriptional Regulators Gene “Silencing” by Modification of Histones and

6、 DNA Eukaryotic Gene Regulation at Steps after Transcription Initiation,Structure of Eukaryotic Regulators Transcriptional Activation Transcriptional Repressors,Structure of Eukaryotic Regulators,The more elaborate transcriptional machinery The nucleosome and their modifiers typical of eukaryotes,Ac

7、tivators/Repressors,Separated DNA Binding and Activation Functions DNA-Binding Domains Activating Regions,Activators Have Separate DNA Binding and Activation Functions,Eukaryotic activators have separate DNA binding and activating regions as well. The two surfaces are very often in separate domains

8、of the protein. (Figure 17-2),FIGURE 17-2 Gal4 bound to its site on DNA.,One such gene is called GAL1. GAL4 binds to four sites located 275bp upstream of GAL1,Gal4 is the most studied eukaryotic activatorGal4 activates transcription of the galactose genes in the yeast S. cerevisae.Gal4 binds to four

9、 sites upstream of GAL1, and activates transcription 1,000-fold in the presence of galactose,Fig; 17-3 The regulatory sequences of the yeast GAL1 gene.,The separate DNA binding and activating domains of Gal4 were revealed in two complementary experiments Expression of the N-terminal region (DNA-bind

10、ing domain) of the activator produces a protein bound to the DNA normally but did not activate transcription. Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor, LexA activates the transcription of the reporter gene. (Domain s),Dom

11、ain s Moving domains among proteins, proving that domains can be dissected into separate parts of the proteins. Many similar experiments shows that DNA binding domains and activating regions are separable.,Bactrial regulatory proteins Most use the helix-turn-helix motif to bind DNA target Most bind

12、as dimers to DNA sequence: each monomer inserts an a helix into the major groove. Eukaryotic regulatory proteins Recognize the DNA using the similar principles, with some variations in detail. Some form heterodimers to recognize DNA, extending the range of DNA-binding specificity.,DNA binding domain

13、s,Homeodomain proteins Zinc containing DNA-binding domain: zinc finger and zinc cluster Leucine zipper motif Helix-Loop-Helix proteins : basic zipper and HLH proteins,Homeodomain proteins: The homeodomain is a class of helix-turn-helix DNA-binding domain and recognizes DNA in essentially the same wa

14、y as those bacterial proteins,Zinc 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 mediated by hydrophobic interactio

15、ns between the appropriately-spaced leucine to form a coiled coil structure,Helix-Loop-Helix motif:,Helix-loop-helix proteins. An extended helical 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 res

16、idues, Leucine zipper and HLH proteins are often called basic zipper and basic HLH proteins.,Both of these proteins use hydrophobic amino acid residues for dimerization.,Activating regions,The activating regions are grouped on the basis of amino acids content Acidic activation domains Glutamine-rich

17、 domains Proline-rich domains,Mechanisms of Eukaryotic Regulators Structure of Eukaryotic Regulators Mechanisms of Eukaryotic Activator Mechanisms of Eukaryotic repressor Signal Transduction and the Control of Transcriptional Regulators Gene “Silencing” by Modification of Histones and DNA Eukaryotic

18、 Gene Regulation at Steps after Transcription Initiation,Mechanisms of Eukaryotic Activator,Eukaryotic activators also work by recruiting as in bacteria, but recruit polymerase indirectly in two ways : 1. Interacting with parts of the transcription machinery. 2. Recruiting nucleosome modifiers that

19、alter chromatin in the vicinity of a gene.,1. Recruitment of the transcription machinery.,The eukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes, such as the Mediator and the TFD complex. Activators interact with one or more of these c

20、omplexes and recruit them to the gene.,TBP in TFIID binds to the TATA box TFIIA and TFIIB are recruited with TFIIB binding to the BRE RNA Pol II-TFIIF complex is then recruited TFIIE and TFIIH then bind upstream of Pol II to form the pre-initiation complex Promoter melting using energy from ATP hydr

21、olysis by TFIIH ) Promoter escapes after the phosphorylation of the CTD tail,Activator Bypass Experiment-Activation of transcription through direct tethering of mediator to DNA.,Directly fuse the bacterial DNA-binding protein LexA protein to the mediator complex Gal11 to activate GAL1 expression.,2.

22、 Activators also recruit Nuleosome modifiers that help the transcription machinery bind at the promoter,Two types of Nucleosome modifiers : Those add chemical groups to the tails of histones, such as histone acetyl transferases (HATs) Those remodel the nucleosomes, such as the ATP-dependent activity

23、 of SWI5/SNF,Local alterations in chromatin directed by activators,Modification of the N-terminal tails of the histones,Modification of the histone N-terminal tails alters the function of chromatin,Fig 7-35 Nucleosome movement catalyzed by nucleosome remodeling complexes,remodling,Effect of histone

24、tail modification,Many enkaryotic activatorsparticularly in higher eukaryoteswork from a distance. Some proteins help, for example Chip protein in Drosophila. The compacted chromosome structure help. DNA is wrapped in nucleosomes in eukaryotes.So sites separated by many base pairs may not be as far

25、apart in the cell as thought.,3. Action at a distance: loops and insulators,Insulators block activation by enhancers,Specific elements called insulators control the actions of activators, preventing the activating the non-specific genes,Transcriptional Silencing Silencing is a specializes form of re

26、pression that can spread along chromatin, switching off multiple genes without the need for each to bear binding sites for specific repressor. Insulator elements can block this spreading, so insulators protect genes from both indiscriminate activation and repression.,4 Appropriate regulation of some

27、 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 genes. Its made up of multiple-sequence elements : something like enhancers, insulators or promoters. It binds

28、regulatory proteins that cause the chromatin structure to “open up”, allowing access to the array of regulators.,Mechanisms of Eukaryotic Regulators Structure of Eukaryotic Regulators Mechanisms of Eukaryotic Activator Mechanisms of Eukaryotic repressor Signal Transduction and the Control of Transcr

29、iptional Regulators Gene “Silencing” by Modification of Histones and DNA Eukaryotic Gene Regulation at Steps after Transcription Initiation, Signal Integration and Combinatorial Control,1. integrate signals,In eukaryotic cells, numerous signals are often required to switch a gene on. So at many gene

30、s multiple activators must work together. They do these by working synergistically: two activators working together is greater than the sum of each of them working alone.,Three strategies of synergy : Two activators recruit a single complex Activators help each other binding cooperativity One activa

31、tor recruit something that helps the second activator bind,a.“Classical” cooperative binding,b. Both proteins interacting with a third protein,c. A protein recruits a remodeller to reveal a binding site for another protein,d. Binding a protein unwinds the DNA from nucleosome a little, revealing the

32、binding site for another protein,Signal integration: the HO gene is controlled by two regulators; one recruits nucleosome modifiers and the other recruits mediator Signal integration: Cooperative binding of activators at the human b-interferon gene.,The examples of integrate signals,The HO gene is i

33、nvolved in the budding of yeast. It has two activators : SWI5 and SBF.,alter the nucleosome,Active only at correct stage of cell cycle,The human -interferon gene is activated in cells upon viral infection. Infection triggers three activators :,NFB, IRF, and Jun/ATF They bind cooperatively to sites w

34、ithin an enhancer, form a structure called Enhanceosome.,Combinatory control lies at the heart of the complexity and diversity of eukaryotes Combinatory control of the mating-type genes from S. cerevisiae,2.Combinatory control,There is extensive combinatorial control in eukaryotes.,In complex multic

35、ellular organisms, combinatorial control involves many more regulators and genes than shown above, and repressors as well as activators can be involved.,Four signals,Three signals,The yeast S.cerevisiae exists in three forms: two haploid cells of different mating types a and a and the diploid formed

36、 when an a and an a cell mate and fuse. Cells 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. cerevisiae,a cell make the regulatory protein a1,a cell make the protein a1 and a2.

37、A fourth regulator 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 yeast,In eukaryotes, repressors dont work by binding to sites that overlap the promoter and thus block binding of polymerase, but mo

38、st common work by recruiting nucleosome modifiers. For example, histone deacetylases repress transcription by removing actetyl groups from the tails of histone.,Transcriptional Repressors,Ways in which eukaryotic repressor Work a and b Ways in which eukaryotic repressor Work c and d,Figure 17-19,In

39、the presence of glucose, Mig1 binds a site between the USAG and the GAL1 promoter. By recruiting the Tup1 repressing complex, Mig1 represses expression of GAL1. Two mechanisms have been proposed to explain the repressing effect of Tup1. First, Tup1 recruits histone deaxetylases. Second, Tup1 interac

40、ts directly with the transcription machinery at the promoter and inhibits initiation.,A specific example: Repression of the GAL1 gene in yeast,Mechanisms of Eukaryotic Regulators Signal Transduction and the Control of Transcriptional Regulators Gene “Silencing” by Modification of Histones and DNA Eu

41、karyotic Gene Regulation at Steps after Transcription Initiation,Structure of Eukaryotic Regulators Transcriptional Activation Transcriptional Repressors,Signal Transduction and the Control of Transcriptional Regulators,1 .Signals are often communicated to transcriptional regulators through signal t

42、ransduction pathway,2 .Signals control the activities of eukaryotic transcriptional regulators in a variety of ways,1.signal transduction involve STAT,a. The STAT pathway,The JAK/STAT Signaling Pathway,2. The MAPK signalling pathways, The RAS-activated MAPK pathway: RAS-RAF-MEK-MAPK represents the f

43、irst 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 ways,Once a signal has been communicated, dir

44、ectly 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 :,Unmasking an activating region Transpor

45、t in or out of the nucleus,Activator Gal4 is regulated by masking protein Gal80,The signalling ligand causes activators (or repressors) to move to the nucleus where they act from cytoplasm.,Mechanisms of Eukaryotic Regulators Signal Transduction and the Control of Transcriptional Regulators Gene “Si

46、lencing” by Modification of Histones and DNA Eukaryotic Gene Regulation at Steps after Transcription Initiation,Gene “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

47、with a dense form of chromatin called heterochromatin. It is frequently associated with particular regions of the chromosome, notably the telomeres, and the centromeres.,Gene “Silencing” by Modification of Histones and DNA,The telomeres, the silent mating-type locus, and the rDNA genes are all “sile

48、nt” regions in S.cerevisiae. Three genes encoding regulators of silencing, SIR2, 3, and 4 have been found (SIR stand for silent information regulator).,Silencing at the yeast telomere,A histone code Different patterns of modification on histone tails can be “read” to mean different things. The “mean

49、ing” would be the result of the direct effects of these modifications on chromatin density and form. But in addition, the particular pattern of modifications at any given location would recruit specific proteins.,Modification of the histone N-terminal tails alters the function of chromatin,Histone m

50、odification and the histone code hypothesis,Transcription can also be silenced by methylation of DNA by enzymes called DNA methylases. This kind of silencing is not found in yeast but is common in mammalian cells. Methylation of DNA sequence can inhibit binding of proteins, including the transcriptional machinery, and thereby block gene expression.,DNA methylases.,Switching a gene off : A mammalian gene marked by methylation of nearby DNA sequence recognized by DNA-binding proteins recruit