CHAPTER 20Regulation of Gene Expression in Eukaryotes.ppt

1、台大農藝系 遺傳學 601 20000,Chapter 20 slide 1,CHAPTER 20 Regulation of Gene Expression in Eukaryotes,Peter J. Russell,edited by Yue-Wen Wang Ph. D. Dept. of Agronomy, NTU,A molecular Approach 2nd Edition,台大農藝系 遺傳學 601 20000,Chapter 20 slide 2,Operons in Eukaryotes,1. It was once believed that eukaryotes do

2、 not have operons, but recent discoveries in nematodes indicate otherwise. Caenorhabditis elegans contains operons as well as typical eukaryotic genes with introns. 2. In nematodes, the operons are controlled from a single promoter, as in prokaryotes. a. Unlike prokaryotes, however, only one protein

3、 can be produced from the mRNA.i. Ribosomes cannot reinitiate at a different start codon on the eukaryotic mRNA.ii. Instead, pre-mRNAs are processed into monogenic mRNAs for individual translation. b. Processing of pre-mRNAs is shown in Figure 20.1:i. RNA polymerase II produces a capped polygenic pr

4、e-mRNA.ii. Cotranslational processing includes transsplicing and generation of the 3 end by cleavage and polyadenylation.iii. Transsplicing using snRNP puts SL-RNA (splice leader) onto the 5 end of a gene in the operon, making the donated SLRNA the leader sequence for each mRNA in the operon.iv. Cle

5、avage and polyadenylation generate 3 ends. c. About 15 percent of C. elegans genes are in operons that range from 28 genes in size.i. Unlike in prokaryotes, no single operon includes all proteins needed for a pathway or a multiprotein complex.ii. Often, genes that work together will be in an operon

6、with genes of unrelated function.,台大農藝系 遺傳學 601 20000,Chapter 20 slide 3,Fig. 20.1 An operon of C. elegans and the production of monogenic mRNAs from a polygenic mRNA by trans-spicing and polyadenylation/cleavage,台大農藝系 遺傳學 601 20000,Chapter 20 slide 4,Levels of Control of Gene Expression in Eukaryot

7、es,1. Prokaryotes respond quickly to their environments mainly by transcriptional (regulatory proteins bind DNA) control. Translational control also occurs, mediated by stability of the mRNAs. 2. Eukaryotes have more complex means to regulate gene expression, because they have compartments (e.g., nu

8、cleus) within cells, and often multicellular structures that require differentiation of cells. 3. Levels at which expression of protein-coding genes is regulated in eukaryotes (Figure 20.2): a. Transcription. b. mRNA processing and transport. c. Translation. d. Degradation of mRNA. e. Protein proces

9、sing. f. Protein degradation.,台大農藝系 遺傳學 601 20000,Chapter 20 slide 5,Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.,Fig. 20.2 Levels at which gene expression can be controlled in eukaryotes,台大農藝系 遺傳學 601 20000,Chapter 20 slide 6,Control of Transcript

10、ion Initiation,1. In eukaryotes, most control of protein gene expression is at the level of transcription initiation, controlled by promoter (immediately upstream) and enhancers (distal from the gene). a. Expression from the promoter alone is at basal level. b. For maximal transcription, activator p

11、roteins bind to:i. Promoter proximal elements.ii. Enhancer elements. 2. Binding of activators: a. Recruits proteins that make the chromatin accessible to the transcription machinery. b. Increases binding of the transcription machinery to the promoter. 3. Variations occur in different genes. .,台大農藝系

12、遺傳學 601 20000,Chapter 20 slide 7,Chromatin Remodeling,1. In eukaryotes, binding of histones to form chromatin generally represses gene expression, making specific repressor proteins unnecessary. 2. Evidence for the role of chromatin structure includes: a. Increased sensitivity to DNaseI of transcrip

13、tionally active genes. b. Hypersensitive DNaseI digestion sites upstream of transcription start sites, corresponding to promoter regions. c. In vitro experiments showing directly that histones can repress gene expression.i. If DNA is simultaneously mixed with both histones and promoter-binding prote

14、ins, it binds more readily to the histones, forming nucleosomes at the TATA box and preventing transcription.ii. If DNA is first mixed with promoter-binding proteins, adding histones does not produce nucleosomes, and transcription occurs.iii. If DNA is simultaneously mixed with histones, binding pro

15、teins: (1) Enhancer-binding proteins bind the enhancer sequences. (2) Promoter-binding proteins bind the promoter sequences. (3) Histones are unable to bind, and so transcription occurs. d. Histones, therefore, are effective repressors, but other proteins can overcome that repression.,台大農藝系 遺傳學 601

16、20000,Chapter 20 slide 8,Activating Genes by Remodeling Chromatin,1. Activation of eukaryotic genes requires alteration off the chromatin structure near the core promoter, a process called chromatin remodeling. Two classes of protein complexes cause chromatin remodeling (Figure 20.3): a. Acetylating

17、 and deacetylating enzymes act on core histones. Histone acetyl transferases (HATs) are part of multiprotein complexes recruited to chromatin when activators bind DNA.i. HATs acetylate lysines in the amino-terminus of core histones.ii. The negative charges of acetyl groups decrease the positive char

18、ges of the histones, reducing their affinity for DNA.iii. Acetylation of histones changes 30-nm chromatin to 10-nm fiber, making promoter more accessible for transcription.iv. The effect is reversible. When histone deacetylases (HDACs) remove acetyl groups, 30-nm chromatin reforms. b. Nucloesome rem

19、odeling complexes (Figure 20.4) are ATP-dependent multiprotein complexes that alter nucleosome positions on the chromatin in response to binding of activators to DNA, increasing transcription. Different types of nucleosome remodeling complexes are known, and some have more than one function:i. Some

20、slide a nucleosome along the DNA, exposing DNA-binding sites for proteins.ii. Some restructure the nucleosome in place.iii. Some transfer the nucleosome from one DNA molecule to another.iv. An example is SWI/SNF, which can remodel using all three methods. Originally discovered in yeasts, where it af

21、fects mating type switch and sucrose fermentation pathways, this complex is now known in many eukaryotes, including mammals.,台大農藝系 遺傳學 601 20000,Chapter 20 slide 9,Fig. 20.3 Chropmatin modeling by (a) histone acetylases and (b) nucleosome remodeling complexes,台大農藝系 遺傳學 601 20000,Chapter 20 slide 10,

22、Fig. 20.4 Activation of transcription by general transcription factors, activators, and a coactivator (“Mediator”),台大農藝系 遺傳學 601 20000,Chapter 20 slide 11,Activation of Transcription by Activators and Coactivators,1. Three classes of proteins are involved in transcription activation: a. General tran

23、scription factors (GTFs), discussed earlier, are required for basal transcription but do not change the rate of transcription initiation. b. Activators (transactivators) are involved in chromatin remodeling to activate transcription.i. There are two key domains, DNA-binding and transcription activat

24、ion, with a flexible region between. Homodimers are often used.ii. Structural motifs for DNA binding regions include (Figure 20.5): (1) Helix-turn-helix (2) Zinc finger (3) Leucine zipperiii. Activation domains are variable. They stimulate transcription initiation up to 100-fold. c. Coactivators are

25、 multiprotein complexes that bind to activators and transcription factors, creating loops in DNA.i. Their presence recruits RNA polymerase II to initiate transcription.ii. Several types of coactivators exist in cells, and their large numbers of proteins make their study difficult.iii. An example of

26、a coactivator is the mediator complex, consisting of 20 or more proteins that bind to activators and to the carboxy-terminal domain of RNA polymerase II.,台大農藝系 遺傳學 601 20000,Chapter 20 slide 12,Fig. 20.5 Examples of the structure motifs (DNA-binding domains) found in DNA-binding proteins such as tra

27、nscription factors and transcription regulator proteins,台大農藝系 遺傳學 601 20000,Chapter 20 slide 13,Blocking Transcription with Repressors,1. Repressors counteract activators for some genes, blocking transcription. a. Two domains occur in repressors, a DNA-binding region and a repressing domain. b. Repr

28、essors work in a variety of ways. Examples:i. Repressor binds near activators binding site, and repressor domain interacts with activation domain of the activator, preventing activation.ii. Repressor binding site overlaps activator binding site, preventing activator binding.iii. Chromatin remodeling

29、 can also block transcription if repressor binds its site and recruits HDAC (histone deacetylase) to cause chromatin compaction.,台大農藝系 遺傳學 601 20000,Chapter 20 slide 14,Combinatorial Gene Regulation,1. Eukaryotic protein-coding gene expression is controlled by: a. Promoters situated just upstream of

30、 the transcription start site.i. Some promoter elements (e.g., TATA) are required to specify the start of transcription, through binding of transcription factor proteins.ii. Regulatory promoter elements are specialized, involving binding by regulatory proteins specific for control of one or a few ge

31、nes.iii. A particular gene may have 1 to many regulatory promoter elements, and one to many regulatory proteins involved in controlling its function.iv. Binding of regulatory proteins to promoters is highly specific to ensure that only the correct genes are activated. b. Enhancers located some dista

32、nce away, either upstream or downstream.i. Enhancers determine whether maximal transcription of the gene occurs.ii. Regulatory proteins bind specific enhancer elements. Which ones bind is determined by the DNA sequence recognized by each protein.iii. Protein interactions determine whether transcript

33、ion is activated or repressed.,台大農藝系 遺傳學 601 20000,Chapter 20 slide 15,2. Promoters and enhancers bind specific regulatory proteins. a. Some regulatory proteins occur in most or all cell types, but others are very specific. b. Each promoter and enhancer has a particular set of proteins that can bind

34、 it, and the combination of proteins bound will determine its expression. c. If both positive and negative regulatory proteins are bound, interactions between them will control the rate of expression. d. When regulatory proteins bind an enhancer and have strong negative effect, the enhancer is a sil

35、encer element. e. Enhancers and promoters appear to bind many of the same proteins, implying interactions of regulatory proteins.i. Relatively few proteins are combined in a variety of ways, regulating the transcription of different arrays of genes.ii. A large number of cell types can be specified b

36、y combinatorial gene regulation.(Figure 20.6),台大農藝系 遺傳學 601 20000,Chapter 20 slide 16,Fig. 20.6 Combinatorial gene regulation,台大農藝系 遺傳學 601 20000,Chapter 20 slide 17,Case Study: Regulation of Galactose Utilization in Yeast,1. Three genes encode enzymes for metabolizing galactose (Figure 20.7): a. GA

37、L1 encodes galactokinase. b. GAL7 encodes galactose transferase. c. GAL10 encodes galactose epimerase. 2. The GAL genes are not transcribed in the absence of galactose, but are rapidly and coordinately induced when galactose is present and glucose is low or absent. Glucose therefore exerts catabolit

38、e repression. a. The GAL genes are near each other, but do not constitute an operon. b. Another nearby gene, GAL4, encodes an activator protein, Gal4p. c. The DNA binding domain of the Gal4p homodimer is a zinc finger that binds the promoter element called an upstream activator sequence-galactose (U

39、ASG).i. Each UASG has four Gal4p binding sites.ii. There is a UASG upstream of the GAL7 gene, and another between the GAL1 and GAL10 genes, which are divergently transcribed from this site. 3. Regulation in response to glucose and galactose also involves the protein Gal80p. a. When galactose is abse

40、nt, a Gal4p dimer binds UASG, along with the repressor protein, Gal80p. No transcription occurs (quenching). b. If galactose is added, its metabolite (produced by Gal3p) binds Gal80p, preventing quenching and allowing Gal4p to activate transcription of GAL7, GAL1, and GAL10. c. Thus:i. Gal4p acts as

41、 a transcriptional activator.ii. Gal80p acts as a repressor.iii. Galactose is an effector molecule.,台大農藝系 遺傳學 601 20000,Chapter 20 slide 18,Fig. 20.7 Regulation of galactose utilization in yeast,台大農藝系 遺傳學 601 20000,Chapter 20 slide 19,Case Study: Regulation of Gene Expression by Hormones,Animation:

42、Regulation of Gene Expression by Steroid Hormones 1. Steroid hormone regulation in animals is another example of short-term control of gene expression. a. Cells of higher eukaryotes perform specialized functions, and are shielded from rapid changes in their environments. One way that a constant envi

43、ronment is maintained in a multicellular organism is by hormone signals.i. Levels of each hormone are maintained by complex feedback loops.ii. Hormones are effector molecules produced by one cell and causing a physiological response in another cell. b. Hormones may deliver their signals in different

44、 ways:i. Some (e.g., steroid hormones) bind cytoplasmic receptors (e.g., steroid hormone receptor, SHR) and then the complex binds directly to DNA, regulating gene expression (Figure 20.8).ii. Others (e.g., polypeptide hormones such as insulin and vasopressin) work at the cell surface by activating

45、a transmembrane enzyme such as adenylate cyclase. The cAMP then acts as a second messenger, transducing the signal to activate cellular events. c. Hormones act only on target cells that have receptors capable of binding the hormone. Receptors for polypeptide hormones are generally on the cell surfac

46、e, while steroid hormone receptors are inside the cell. d. Steroid hormones are well studied. All have a common four-ring structure, and physiological effects derive from the differences in side groups (Figure 20.9).,台大農藝系 遺傳學 601 20000,Chapter 20 slide 20,Fig. 20.8 Mechanisms of action of polypepti

47、de hormones and steroid hormones,台大農藝系 遺傳學 601 20000,Chapter 20 slide 21,Fig. 20.9 Structure of some mammalian steroid hormones,台大農藝系 遺傳學 601 20000,Chapter 20 slide 22,e. Steroid hormones show tissue-specific effects. Examples (Table 20.1):i. Estrogen induces prolactin in rat pituitary, vitellogenin

48、 in frog liver, and conalbumin, lysozyme, ovalbumin, and ovomucoid in the hen oviduct.ii. Glucocorticoids induce synthesis of growth hormone in rat pituitary, and phosphoenolpyruvate carboxykinase in rat kidney. f. Hormone receptors control the specificity of the response, because only cells with re

49、ceptors can detect and respond to the hormone.i. Steroid hormones affect transcription and stability of mRNAs, and possibly processing of mRNA precursors.ii. SHRs have high affinity for their respective hormones, and all work in the same way. (1) If the hormone is absent, its SHR is found associated

50、 with chaperone proteins, including Hsp90. The SHR is inactive. (2) When hormone enters the cell, it binds its specific SHR, displacing Hsp90 and forming a glucocorticoid-SHR complex (Figure 20.10). (3) When steroid hormone binds SHR, the complex is found in the nucleus, where it binds specific DNA regulatory sequences, often by a zinc finger domain, activating or inactivating transcription of genes controlled by the hormone.,