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Regulation of Gene Expression 基因表达调控 Deqiao Sheng PhD Dept. of Biochemistry and Molecular Biology.

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Presentation on theme: "Regulation of Gene Expression 基因表达调控 Deqiao Sheng PhD Dept. of Biochemistry and Molecular Biology."— Presentation transcript:

1 Regulation of Gene Expression 基因表达调控 Deqiao Sheng PhD Dept. of Biochemistry and Molecular Biology

2 Reference Books 1.Leningers’ Principles of Biochemistry 2.Harpers’ Biochemistry 26th edition 3.Styers’ Biochemistry 4.Hortons’ Principles of Biochemistry 4th edition

3 Diagram of the central dogma, DNA to RNA to protein, illustrating the genetic code. Gene expression Basic conceptions

4 A gene( 基因 ) is the basic unit of heredity in a living organism. All living things depend on genes. Genes hold the information to build and maintain an organism's cells and pass genetic traits to offspring. Function units

5 Genome —— is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. E.coli contains about 4,400 genes present on a single chromosome Human genome is more complex, with 23 pairs of chromosomes containing 6 billion(6×10 9 ) base pairs of DNA. 30,000~40,000 genes

6 Concept of gene expression Gene expression is the combined process of the transcription of a gene into mRNA, the processing of that mRNA, and its translation into protein Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is a functional RNA.

7 The genetic information present in each somatic cell of a metazoan organism (multicellular animals ) is practically identical. How to meet the different needs? Different function need different proteins. Regulated expression of genes is required for development, differentiation, and adaption.

8 In genetics gene expression is the most fundamental level at which genotype gives rise to the phenotype. The genetic code is "interpreted" by gene expression, and the properties of the expression products give rise to the organism's phenotype. Genotype→Phenotype

9 Information Flow A gene is turned on and transcribed into RNA A gene is turned on and transcribed into RNA Information flows from genes to proteins, genotype to phenotype Information flows from genes to proteins, genotype to phenotype Genotype Phenotype

10 The cellular concentration of a protein is determined by a delicate balance of at least seven processes, each having several potential points of regulation.

11 Points of Regulation 1.Transcription 2.Post-transcriptional modification 3.mRNA degradation rate 4.Translation 5.Post-translational modification 6.Protein targeting and transport 7.Protein degradation

12 Regulation of Gene Expression General The regulation of the expression of genes is absolutely essential for the growth, development, differentiation and the very existence of an organism. The are two types of gene regulation- positive and negative.

13 Positive regulation: the gene regulation is said to be positive when its expression is increased by a regulatory element (positive regulator) Negative regulation: A decrease in the gene expression due to the presence of a regulatory element (negative regulator) is referred to as negative regulation.

14 The aim of the control What When Where To select the right gene To express at the right time To express at the right place The right gene expresses at the right time & the right place.

15 The significance of gene expression regulation The differential transcription of different genes largely determines the actions and properties of cells. Regulation at any one of the various steps in this process could lead to differential gene expression in different cell types or developmental stages or in response to external conditions (such as: Environments). Temporal specificity (stage specificity) Spatial specificity (cell or tissue specificity)

16 Four of the many different types of human cells –They all share the same genome Genotype (DNA) –What makes them different? Phenotype (Protein) FROM EGG TO ORGANISM: HOW AND WHY GENES ARE REGULATED (a) Three muscle cells (partial)(b) A nerve cell (partial) (c) Sperm cells(d) Blood cells One of underlying principles of molecular cell biology is that the actions and properties of each cell type are determined by the proteins it contains.

17 One of the characters of gene expression : it is precisely controlled to be activated in the right cells and right time during development of the many different cell types that collectively form a multicellular organism. e.g. Human Hemoglobin( 血红蛋白 ) Human hemoglobin is consisted of two alpha-like and beta-like globin chains, which are coded by alpha-like and beta-like globin genes respectively.

18 Hemoglobin clusters Human hemoglobin: (at developmental stages)  2  2 HbF  2  2 (end of trimester) HbA  2  2 (start from the third trimester, do not completely replace  chains until some weeks postpartum)

19 Regulation of Gene Expression 1. Principles of gene regulation 2. Regulation of gene expression in prokaryotes 3. Regulation of gene expression in eukaryotes

20 Principles of Gene Regulation

21 1. Constitutive gene expression A gene is expressed at the same level at all times. housekeeping gene 2. Regulated gene expression Inducible :Gene products that increase in concentration under particular molecular circumstances. Repressible: gene products that decrease in concentration in response to a molecular signal.

22 Constitutive genes Constitutive genes: refer to genes whose expression are not regulated. The products of these genes are produced at a constant rate. Such genes are called constitutive genes and their expression is said to be constitutive. e.g.  -actin-Actins are highly conserved proteins that are involved in cell motility, structure and integrity. GAPDH ( glyceraldehyde-3-phosphate dehydrogenase )- is an enzyme that catalyzes the sixth step of glycolysis and thus serves to break down glucose for energy and carbon molecules.

23 Products of the constitutive genes are required at all times, such as those for the enzymes of central metabolic pathways. Those genes are expressed at a more or less constant level in virtually every cell of a species or organism. They are often referred to as housekeeping genes also.

24 Inducible genes Inducible genes refer to the genes whose expression increases in response to an inducer, a specific regulatory signal. The process is called induction. e.g. The expression of many of the genes encoding DNA repair enzymes, for example, is induced by high levels of DNA damage.

25 The Structure of Gene Structural gene codes for a protein (or RNA) product Regulatory gene codes for a protein (or an RNA) involved in regulating the expression of other genes

26 Structural and Regulatory gene A structural gene : Structural genes represent an enormous variety of protein structures and functions, including structural proteins, enzymes and regulatory proteins. A regulatory gene : The interaction can regulate a target gene in a manner either positive (the interaction turns the gene on) or negative (the interaction turns the gene off).

27 RNA Polymerase Binds to DNA at Promoters RNA polymerases bind to DNA and initiate transcription at promoters, sites generally found near points at which RNA synthesis begins on the DNA template. The nucleotide sequences of promoters vary considerably, affecting the binding affinity of RNA polymerases and thus the frequency of transcription initiation.

28 Consensus sequence for many E. coli promoters. (procaryotic) -10 region TATAAT -35 region TTGACA Most base substitutions in the -10 and -35 regions have a negative effect on promoter function. Some promoters also include the UP (upstream promoter) element Sequences of promoters

29 Transcription activity ? promoter sequence Mutations that result in a shift away from the consensus sequence usually decrease promoter function; conversely, mutations toward consensus usually enhance promoter function. regulatory proteins It can modulate non-housekeeping genes expression

30 Common sequences in promoters recognized by eukaryotic RNA polymerase II. -30 region TATA box Initiator sequence (Inr) N, represents any nucleotide Y, a pyrimidine nucleotide

31 RNA Polymerase II Requires Many Other Protein Factors for Its Activity 1.specificity factors –Alter the specificity of RNA polymerase for a given promoter or set of promoters 2.repressors –impede access of RNA polymerase to the promoter 3.activators –Enhance the RNA polymerase–promoter interaction.

32 RNA polymerase II holoenzyme complex bound to a promoter Transcription machinery

33 There are a lot of proteins participate in the regulation of gene expression. –Transcription Factor (TF) –Activators –Repressors –Regulatory proteins

34 Specificity factors Prokaryotic specificity factors –The  subunit of the E. coli RNA polymerase holoenzyme is a specificity factor that mediates promoter recognition and binding. Eukaryotic specificity factors –the TATA-binding protein (TBP)

35 Repressors Protein  Bind to specific sites on the DNA  In prokaryotic cells, such binding sites, called operators, are generally near a promoter.  Blocks transcription/negative regulation

36 Activators Activators provide a molecular counterpoint to repressors; they bind to DNA and enhance the activity of RNA polymerase at a promoter  positive regulation  binding sites are often adjacent to promoters that are bound weakly or not at all by RNA polymerase alone

37 Enhancers positive regulation Some eukaryotic activators bind to DNA sites, called enhancers, that are quite distant from the promoter, affecting the rate of transcription at a promoter that may be located thousands of base pairs away. Some activators are normally bound to DNA, enhancing transcription until dissociation of the activator is triggered by the binding of a signal molecule.

38 Regulatory proteins Three domain (at least two) 1. DNA binding domain Bind to DNA 2. protein-protein interaction domain Interact with RNA polymerase, other regulatory proteins, or other subunits of the same regulatory protein. 3. dimerization domain Domain—An independently folded part of a protein.

39 Within regulatory proteins, the amino acid side chains most often hydrogen-bonding to bases in the DNA are those of Asn, Gln, Glu, Lys, and Arg residues. To interact with bases in the major groove of DNA, a protein requires a relatively small structure that can stably protrude from the protein surface.

40 DNA-binding sites The DNA-binding sites for regulatory proteins are often inverted repeats of a short DNA sequence (a palindrome) at which multiple (usually two) subunits of a regulatory protein bind cooperatively. The Lac repressor is unusual in that it functions as a tetramer, with two dimers tethered together at the end distant from the DNA-binding sites.

41 Relationship between the lac operator sequence and the lac promoter. palindrome AATTGT…ACAATT TTAACA…TGTTAA

42 DNA binding domain : DNA-binding sites :a short DNA sequence (a palindrome)  helix-turn-helix  zinc finger  homeodomain—found in some eukaryotic proteins.

43 helix-turn-helix This DNA-binding motif is crucial to the interaction of many prokaryotic regulatory proteins with DNA, and similar motifs occur in some eukaryotic regulatory proteins. The helix-turn-helix motif comprises about 20 amino acids in two short  -helical segments, each seven to nine amino acid residues long, separated by a  turn

44 Helix-turn-helix DNA-binding domain of the Lac repressor. The helix-turn-helix motif is shown in red and orange; the DNA recognition helix is red.

45 Zinc Finger In a zinc finger, about 30 amino acid residues form an elongated loop held together at the base by a single Zn 2+ ion, which is coordinated to four of the residues (four Cys, or two Cys and two His). The zinc does not itself interact with DNA; rather, the coordination of zinc with the amino acid residues stabilizes this small structural motif. Several hydrophobic side chains in the core of the structure also lend stability.

46 Zinc fingers. Three zinc fingers (gray) of the regulatory protein Zif268, complexed with DNA (blue and white). Each Zn 2+ (maroon) coordinates with two His and two Cys residues (not shown).

47 Homeodomain Another type of DNA-binding domain has been identified in a number of proteins that function as transcriptional regulators, especially during eukaryotic development. This domain of 60 amino acids—called the homeodomain, because it was discovered in homeotic genes (genes that regulate the development of body patterns)—is highly conserved and has now been identified in proteins from a wide variety of organisms, including humans. The DNA-binding segment of the domain is related to the helix-turn-helix motif. The DNA sequence that encodes this domain is known as the homeobox.

48 Homeodomain. Shown here is a homeodomain bound to DNA; one of the helices (red), stacked on two others, can be seen protruding into the major groove. This is only a small part of the much larger protein Ultrabithorax (Ubx), active in the regulation of development in fruit flies.

49 Motif— An independent folding unit, or particular structure, that recurs in many molecules. (DNA or protein) Domain— An independently folded part of a protein.

50 protein-protein interaction domain: Mediate interaction with RNA polymerase, other regulatory proteins, or other subunits of the same regulatory protein. –leucine zipper –basic helix-loop-helix.

51 Leucine Zipper This motif is an amphipathic helix with a series of hydrophobic amino acid residues concentrated on one side, with the hydrophobic surface forming the area of contact between the two polypeptides of a dimer. A striking feature of these helices is the occurrence of Leu residues at every seventh position, forming a straight line along the hydrophobic surface. Although researchers initially thought the Leu residues interdigitated (hence the name “zipper”)

52 Leucine zippers (a) Comparison of amino acid sequences of several leucine zipper proteins. Note the Leu (L) residues at every seventh position in the zipper region, and the number of Lys (K) and Arg (R) residues in the DNA-binding region. (b) Leucine zipper from the yeast activator protein GCN4 (PDB ID 1YSA). Only the “zippered” helices (gray and light blue), derived from different subunits of the dimeric protein, are shown. The two helices wrap around each other in a gently coiled coil. The interacting Leu residues are shown in red.

53 Basic Helix-Loop-Helix (bHLH) Another common structural motif occurs in some eukaryotic regulatory proteins implicated in the control of gene expression during the development of multicellular organisms. These proteins share a conserved region of about 50 amino acid residues important in both DNA binding and protein dimerization. This region can form two short amphipathic  helices linked by a loop of variable length, the helix- loop-helix.

54 distinct from the helix-turn-helix (motif associated with DNA binding) The helix-loop-helix motifs of two polypeptides interact to form dimers. In these proteins, DNA binding is mediated by an adjacent short amino acid sequence rich in basic residues, similar to the separate DNA-binding region in proteins containing leucine zippers.

55 Helix-loop-helix. The human transcription factor Max, bound to its DNA target site. The protein is dimeric; one subunit is colored. The DNA-binding segment (pink) merges with the first helix of the helix-loop-helix (red). The second helix merges with the carboxyl-terminal end of the subunit (purple). Interaction of the carboxyl- terminal helices of the two subunits describes a coiled coil very similar to that of a leucine zipper, but with only one pair of interacting Leu residues (red side chains near the top) in this particular example. The overall structure is sometimes called a helix-loop-helix/leucine zipper motif. DNA-binding carboxyl-terminal end

56 SUMMARY 1.The expression of genes is regulated by processes that affect the rates at which gene products are synthesized and degraded. Much of this regulation occurs at the level of transcription initiation, mediated by regulatory proteins that either repress transcription (negative regulation) or activate transcription (positive regulation) at specific promoters.

57 2.Regulatory proteins are DNA-binding proteins that recognize specific DNA sequences; most have distinct DNA- binding domains. Within these domains, common structural motifs that bind DNA are the helix-turn-helix, zinc finger, and homeodomain.

58 3.Regulatory proteins also contain domains for protein-protein interactions, including the leucine zipper and helix-loop-helix, which are involved in dimerization, and other motifs involved in activation of transcription.

59 Regulation of gene expression in prokaryotes

60 Structure of Prokaryote Genome is smaller than eukaryotes No nucleus (DNA and a few associated pro.) nucleoid Gene cluster Transcription and translation are coupled Polycistrons

61 Prokaryotes Provide Models for the Study of Gene Expression in Mammalian Cells Regulation at two levels Transcriptional regulation Post-transcriptional regulation Operon model Two well-studied operons: lac operon trp operon

62 François Jacob (1920 – ). Jacques Monod (1910–1976). Jacob and Monod received the Nobel Prize in Physiology or Medicine in 1965 for their work on the genetic control of enzyme synthesis. The concept of operon was introduced by Jacob and Monod in 1961

63 Many Prokaryotic Genes Are Clustered and Regulated in Operons Many prokaryotic mRNAs are polycistronic— multiple genes on a single transcript—and the single promoter that initiates transcription of the cluster is the site of regulation for expression of all the genes in the cluster. The gene cluster and promoter, plus additional sequences that function together in regulation, are called an operon

64 Operon- is the coordinated unit of genetic expression in bacteria. It is an operator plus two or more genes under control of that operator. Occurs only in prokaryotes (in eukaryotes, each gene is under separate control). Best known is the lac operon

65 Promoter (P) RNA pol control sequence site where the transcription enzyme initiates transcription Operator (O) Repressor Is a DNA sequence between the promoter and the enzyme genes Acts as an on and off switch for the genes Structural Genes One to several genes coding for enzymes of a metabolic pathway Translated simultaneously as a block The Structure of lactose operon

66 OP Representative prokaryotic operon Genes A, B, and C are transcribed on one polycistronic mRNA. Typical regulatory sequences include binding sites for proteins that either activate or repress transcription from the promoter.

67 Operons—the basic concept of Prokaryotic Gene Regulation Regulated genes can be switched on and off depending on the cell’s metabolic needs. Operon-a regulated cluster of adjacent structural genes, operator site, promotor site, and regulatory gene(s).

68 The lac Operon Is Subject to Negative Regulation The study of gene regulation began with the lactose operon in E.coli. The operon model was proposed to explain the regulation of RNA synthesis related to lactose metabolism in E.coli First introduced the concept of operon, operator, repressor, inducer in gene regulation. Jacob and Monod in 1961 described their operon model in a classic paper.

69 The structure of Lac Operon structural gene  β-galactosidase (lacZ),  galactoside permease(lacY)  thiogalactoside transacetylase (lacA). regulatory gene  lac promoter P  lac operator O

70 lacZ, lacY, lacA P-Promoter, O-Operator I-Inhibitor

71 The roles of the three structural genes lacZ codes for the enzyme β-galactosidase, whose active form is a tetramer of ~500 kD. The enzyme breaks a β-galactoside into its component sugars. lacY codes for the β-galactoside permease, a 30 kD membrane-bound protein constituent of the transport system. This transports β -galactosides into the cell. lacA codes for β-galactoside transacetylase, an enzyme that transfers an acetyl group from acetyl-CoA to β-galactosides.

72 Lactose metabolism in E. coli. β β Uptake and metabolism of lactose require the activities of galactoside permease and β- galactosidase. Conversion of lactose to allolactose by transglycosylation is a minor reaction also catalyzed by β- galactosidas

73 Each of these linked genes is transcribed into one large mRNA molecule that contains multiple independent translation start (AUG) and stop (UAA) codons for each cistron. Thus, each protein is translated separately, and they are not processed from a single large precursor protein. This type of mRNA molecule is called a polycistronic mRNA.

74 How to write a gene and a protein? A gene is generally italicized in lower case and the encoded protein, when abbreviated, is expressed in roman type with the first letter capitalized. –For example, the gene lacI encodes the repressor protein LacI.

75 Off On

76 Several -galactosides structurally related to allolactose are inducers of the lac operon but are not substrates for -galactosidase; others are substrates but not inducers. One particularly effective and nonmetabolizable inducer of the lac operon that is often used experimentally is isopropylthiogalactoside (IPTG):

77 The lac Operon Undergoes Positive Regulation The operator-repressor-inducer interactions described earlier for the lac operon provide an intuitively satisfying model for an on/off switch in the regulation of gene expression. Operon regulation is rarely so simple Glucose affect the expression of the lac genes

78 Glucose, metabolized directly by glycolysis, is E. coli’s preferred energy source. Other sugars can serve as the main or sole nutrient, but extra steps are required to prepare them for entry into glycolysis, necessitating the synthesis of additional enzymes. Clearly, expressing the genes for proteins that metabolize sugars such as lactose or arabinose is wasteful when glucose is abundant.

79 What happens to the expression of the lac operon when both glucose and lactose are present? A regulatory mechanism known as catabolite repression restricts expression of the genes required for catabolism of lactose, arabinose, and other sugars in the presence of glucose, even when these secondary sugars are also present. The effect of glucose is mediated by cAMP, as a coactivator, and an activator protein known as cAMP receptor protein, or CRP (the protein is sometimes called CAP, for catabolite gene activator protein).

80 CRP is a homodimer with binding sites for DNA and cAMP. Binding is mediated by a helix-turn- helix motif within the protein’s DNA-binding domain. When glucose is absent, CRP-cAMP binds near the lac promoter and stimulates RNA to a site transcription 50-fold. CRP-cAMP is therefore a positive regulatory element responsive to glucose levels, whereas the Lac repressor is a negative regulatory element responsive to lactose.

81 Activation of transcription of the lac operon by CRP Sequence of the lac promoter compared with the promoter consensus sequence. The differences mean that RNA polymerase binds relatively weakly to the lac promoter until the polymerase is activated by CRP-cAMP.

82 Combined effects of glucose and lactose on expression of the lac operon. (a) High levels of transcription take place only when glucose concentrations are low (so cAMP levels are high and CRP-cAMP is bound) and lactose concentrations are high (so the Lac repressor is not bound). (b) Without bound activator (CRP-cAMP), the lac promoter is poorly transcribed even when lactose concentrations are high and the Lac repressor is not bound. Off On

83 Regulation of transcription from the lac operon of E.coli.

84 Levels of Control of Lac Operon Expression 3 Scenarios: 1.No Lactose around –Operon switched off, no mRNA regardless of [glucose] 2.Lactose present; glucose also present –The presence of lactose inactivates the repressor –  Transcription occurs –Glucose present  cAMP is low  CRP does not ‘help’ transcription 3.Lactose present; no glucose –The presence of lactose inactivates the repressor –  Transcription occurs –NO Glucose  cAMP is high  cAMP binds CRP (becomes activated)  CRP binds & ‘Helps’ Transcription –High Level of transcription

85 Many Genes for Amino Acid Biosynthetic Enzymes Are Regulated by Transcription Attenuation The genes for the enzymes needed to synthesize a given amino acid are generally clustered in an operon and are expressed whenever existing supplies of that amino acid are inadequate for cellular requirements. When the amino acid is abundant, the biosynthetic enzymes are not needed and the operon is repressed.

86 trp operon The E. coli tryptophan (trp) operon includes five genes for the enzymes required to convert chorismate to tryptophan. Note that two of the enzymes catalyze more than one step in the pathway. The mRNA from the trp operon has a half-life of only about 3 min, allowing the cell to respond rapidly to changing needs for this amino acid. The Trp repressor is a homodimer, each subunit containing 107 amino acid residues.

87 The trp operon

88 When tryptophan is abundant it binds to the Trp repressor, causing a conformational change that permits the repressor to bind to the trp operator and inhibit expression of the trp operon. The trp operator site overlaps the promoter, so binding of the repressor blocks binding of RNA polymerase. This simple on/off circuit mediated by a repressor is not the entire regulatory story.

89 Transcription attenuation mechanism Different cellular concentrations of tryptophan can vary the rate of synthesis of the biosynthetic enzymes over a 700-fold range. Once repression is lifted and transcription begins, the rate of transcription is fine-tuned by a second regulatory process, called transcription attenuation, in which transcription is initiated normally but is abruptly halted before the operon genes are transcribed. The frequency with which transcription is attenuated is regulated by the availability of tryptophan and relies on the very close coupling of transcription and translation in bacteria.


91 The trp operon attenuation mechanism uses signals encoded in four sequences within a 162 nucleotide leader region at the 5 end of the mRNA, preceding the initiation codon of the first gene. Within the leader lies a region known as the attenuator, made up of sequences 3 and 4.

92 Transcriptional attenuation in the trp operon. Transcription is initiated at the beginning of the 162 nucleotide mRNA leader encoded by a DNA region called trpL. A regulatory mechanism determines whether transcription is attenuated at the end of the leader or continues into the structural genes.


94 Regulation of gene expression in eukaryotes

95 Structure of Eukaryote Genome is bigger Nucleus (DNA and histone, nucleosome, chromatin) Transcription and translation is separated. Post-transcriptional modification Split gene (Exon and Intron)

96 Each cell of the higher organisms contains the entire genome. Gene expression in eukaryotes is regulated to provide the appropriate response to biological needs. 1. Expression of certain genes (housekeeping gene) in most of cells. 2. Activation of selected genes upon demand. 3. Permanent inactivation of several genes in all but a few types.

97 In case of prokaryotic cells, most of the DNA is organized into genes which can be transcribed. In contrast, in mammals, very little of the total DNA is organized into genes and their associated regulatory sequence. Eukaryotic gene expression and its regulation are highly complex!!!

98 Four important features of the regulation of gene expression in eukaryotes First, access to eukaryotic promoters is restricted by the structure of chromatin, and activation of transcription is associated with many changes in chromatin structure in the transcribed region. Second, although eukaryotic cells have both positive and negative regulatory mechanisms, positive mechanisms predominate in all systems characterized so far. Thus, given that the transcriptional ground state is restrictive, virtually every eukaryotic gene requires activation to be transcribed.

99 Third, eukaryotic cells have larger, more complex multimeric regulatory proteins than do bacteria. Finally, transcription in the eukaryotic nucleus is separated from translation in the cytoplasm in both space and time.

100 The Greater Complexity of Eukaryotic Genomes Requires Elaborate Mechanisms for Gene Regulation Gene regulation is significantly more complex in eukaryotes than in prokaryotes for a number of reasons.

101 First, the genome being regulated is significantly larger. –The E. coli genome consists of a single, circular chromosome containing 4.6 Mb. This genome encodes approximately 2000 proteins. – In comparison, one of the simplest eukaryotes, Saccharomyces cerevisiae (baker's yeast), contains 16 chromosomes ranging in size from 0.2 to 2.2 Mb. The yeast genome totals 17 Mb and encodes approximately 6000 proteins. – The genome within a human cell contains 23 pairs of chromosomes ranging in size from 50 to 250 Mb. Approximately 40,000 genes are present within the 3000 Mb of human DNA.

102 It would be very difficult for a DNA-binding protein to recognize a unique site in this vast array of DNA sequences. Consequently, more- elaborate mechanisms are required to achieve specificity. Another source of complexity in eukaryotic gene regulation is the many different cell types present in most eukaryotes. (differentiation) –Liver and pancreatic cells, for example, differ dramatically in the genes that are highly expressed.

103 Moreover, eukaryotic genes are not generally organized into operons. Instead, genes that encode proteins for steps within a given pathway are often spread widely across the genome. Finally, transcription and translation are uncoupled in eukaryotes, eliminating some potential gene-regulatory mechanisms.

104 Chromatin structure and gene expression The DNA in higher organisms is extensively folded and packed to form protein-DNA complex called chromatin. The structural organization of DNA in the form of chromatin plays an important role in eukaryotic gene expression.

105 Condense Decondense

106 Transcriptionally Active Chromatin Is Structurally Distinct from Inactive Chromatin Several forms of chromatin can be found : About 10% of the chromatin in a typical eukaryotic cell is in a more condensed form than the rest of the chromatin. This form, heterochromatin, is transcriptionally inactive. Heterochromatin is generally associated with particular chromosome structures—the centromeres, for example. The remaining, less condensed chromatin is called euchromatin.

107 Transcription of a eukaryotic gene is strongly repressed when its DNA is condensed within heterochromatin. Some, but not all, of the euchromatin is transcriptionally active.

108 Types of Chromatin Heterochromatin highly condensed during interphase, not actively transcribed Euchromatin less condensed during interphase, able to be transcribed

109 Nucleosomes Are Complexes of DNA and Histones The DNA in eukaryotic chromosomes is not bare. Rather eukaryotic DNA is tightly bound to a group of small basic proteins called histones. Five major histones are present in chromatin: H2A, H2B, H3, and H4 (histone octamer) H1 Histones have strikingly basic properties because a quarter of the residues in each histone is either arginine or lysine The entire complex of a cell's DNA and associated protein is called chromatin.

110 In 1974, Roger Kornberg proposed that chromatin is made up of repeating units, each containing 200 bp of DNA and two copies each of H2A, H2B, H3, and H4, called the histone octamer. These repeating units are known as nucleosomes. This smaller complex of the histone octamer and the 145-bp DNA fragment is the nucleosome core particle. The DNA connecting core particles in undigested chromatin is called linker DNA. Histone H1 binds, in part, to the linker nDNA.

111 Levels of Chromatin Structure

112 Eukaryotic DNA Is Wrapped Around Histones to Form Nucleosomes The eight histones in the core are arranged into a (H3) 2 (H4) 2 tetramer and a pair of H2A/H2B dimers. The tetramer and dimers come together to form a left- handed superhelical ramp around which the DNA wraps.

113 Each histone has an amino-terminal tail that extends out from the core structure. These tails are flexible and contain a number of lysine and arginine residues. As we shall see, covalent modifications of these tails play an essential role in modulating the affinity of the histones for DNA and other properties.


115 Histone modifications Phosphorylaiton Acetylation Methylation Ubiqitination SUMOlation ADP-ribosylation

116 The acetylation and deacetylation of histones figure prominently in the processes that activate chromatin for transcription. As noted above, the amino-terminal domains of the core histones are generally rich in Lys residues. Particular Lys residues are acetylated by histone acetyltransferases (HATs). Where chromatin is being activated for transcription, the nucleosomal histones are further acetylated by nuclear HATs.

117 Enhancers Can Stimulate Transcription by Perturbing Chromatin Structure Enhancer, DNA sequences, although they have no promoter activity of their own, greatly increase the activities of many promoters in eukaryotes, even when the enhancers are located at a distance of several thousand base pairs from the gene being expressed. Enhancers function by serving as binding sites for specific regulatory proteins. An enhancer is effective only in the specific cell types in which appropriate regulatory proteins are expressed.

118 In many cases, these DNA-binding proteins influence transcription initiation by perturbing the local chromatin structure to expose a gene or its regulatory sites rather than by direct interactions with RNA polymerase. This mechanism accounts for the ability of enhancers to act at a distance.

119 Many Eukaryotic Promoters Are Positively Regulated Positive regulation?—— The storage of DNA within chromatin effectively renders most promoters inaccessible, so genes are normally silent in the absence of other regulation. The structure of chromatin affects access to some promoters more than others, but repressors that bind to DNA so as to preclude access of RNA polymerase (negative regulation) would often be simply redundant.

120 DNA-Binding Transactivators and Coactivators Facilitate Assembly of the General Transcription Factors Successful binding of active RNA polymerase II holoenzyme at one of its promoters usually requires the action of other proteins : 1.basal transcription factors, required at every Pol II promoter; 2.DNA binding transactivators, which bind to enhancers or UASs and facilitate transcription; and 3.coactivators. The latter group act indirectly—not by binding to the DNA—and are required for essential communication between the DNA-binding transactivators and the complexcomposed of Pol II and the general transcription factors.

121 TBP--TATA-binding protein Protein—protein interaction!

122 Eukaryotic Gene Expression Can Be Regulated by Intercellular and Intracellular Signals Regulation Can Result from Phosphorylation of Nuclear Transcription Factors Many Eukaryotic mRNAs Are Subject to Translational Repression Development Is Controlled by Cascades of Regulatory Proteins

123 SUMMARY 1.The expression of genes is regulated by processes that affect the rates at which gene products are synthesized and degraded. Much of this regulation occurs at the level of transcription initiation, mediated by regulatory proteins that either repress transcription (negative regulation) or activate transcription (positive regulation) at specific promoters.

124 2.In bacteria, genes that encode products with interdependent functions are often clustered in an operon, a single transcriptional unit. Transcription of the genes is generally blocked by binding of a specific repressor protein at a DNA site called an operator. Dissociation of the repressor from the operator is mediated by a specific small molecule, an inducer. These principles were first elucidated in studies of the lactose (lac) operon. The Lac repressor dissociates from the lac operator when the repressor binds to its inducer, allolactose.

125 3.Regulatory proteins are DNA-binding proteins that recognize specific DNA sequences; most have distinct DNA- binding domains. Within these domains, common structural motifs that bind DNA are the helix-turn-helix, zinc finger, and homeodomain.

126 4.Regulatory proteins also contain domains for protein-protein interactions, including the leucine zipper and helix-loop-helix, which are involved in dimerization, and other motifs involved in activation of transcription.

127 5.In eukaryotes, positive regulation is more common than negative regulation, and transcription is accompanied by large changes in chromatin structure. Promoters for Pol II typically have a TATA box and Inr sequence, as well as multiple binding sites for DNA-binding transactivators.

128 Operon Structural gene--gene that codes for a polypeptide Promoter region--controls access to the structural genes, located between the promoter and structural genes, contains the operator site. Operator Site--region where the repressor attaches Regulatory genes--codes for repressor proteins Polycistronic mRNA--transcript for several polypeptides

129 REVIEW QUESTIONS For each question, choose the ONE BEST answer The lac operon is transcribed when A. lactose is present and glucose is absent. B. cAMP concentrations in the cell are high. C. The cAMP–CAP protein is bound to the lac promoter region. D. the lac repressor is bound to allolactose or a similar shaped molecule. E. all of the above.

130 How lac operon works? –Negative regulation –Positive regulation

131 Key Terms 1.Gene 2.Gene expression 3.Housekeeping gene 4.Operon 5.Operator 6.Promoter 7.Enhancer

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