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MCB 317 Genetics and Genomics Topic 9 Overview of Eukaryotic Gene Expression.

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Presentation on theme: "MCB 317 Genetics and Genomics Topic 9 Overview of Eukaryotic Gene Expression."— Presentation transcript:

1 MCB 317 Genetics and Genomics Topic 9 Overview of Eukaryotic Gene Expression

2 Gene Regulation in Eukaryotes

3 Readings Chromatin: Hartwell Chapter 12, pages Heterochromatin: Hartwell Chapter 12, section 12.3

4 Concept: Every step in a biological process is a potential site of regulation Gene Expression v. Transcription

5 Outline Txn in Prokaryotes Overview of Txn in Eukaryotes DNA Binding Proteins (“Txn Factors”) Chromatin 1. Knowledge / Facts / Language 2. Knowing HOW we know what we know 3. Asking new questions & discovering answers

6 Expectations and Review 1.Prokaryotes: Basic process and nomenclature Process of txn Start and stop signals for txn Gene orientation One RNAP

7 Expectations and Review Nomenclature: ORF, promoter, codon, Start/stop codons, mRNA, untranslated region, tRNA, consensus sequence, homolog, coding and noncoding strands, activator (proteins), repressor, etc…

8 Consensus sequence Prokaryotes

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10 Lodish 11-9 Why consensus and not exact sequence? How Does RNAP “find” its Promoter and Initiate Txn? TATA(A/T)A(A/T)(A/G)

11 Consensus sequences provide for binding to specific DNA sites over a range of affinities Concept: Biological Reactions are often Optimized, not Maximized

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13 Synthesis/polymerization is in the 5’ to 3’ direction

14 Coding v. non-coding strand, Directionality Coding looks like mRNA Non-coding can base-pair With mRNA

15 Promoters are Directional

16 Activators

17 Repressors

18 Concept: Turning Genes ON and OFF ON -> Activated OFF -> Repressed OFF -> Not Activated In General: Repressors Win

19 Distinguishing: Activators from Repressors Positive Regulators from Negative Regulators Key: What is the role of the active form of the protein

20 Regulation: Activation/Repression in Response to Particular Conditions

21 Repressors Activators and Repressors vs. Inducers

22 Outline Txn in Prokaryotes (Review) Overview of Txn in Eukaryotes Chromatin

23 Thinking About Prokaryotic v. Eukaryotic Txn 1. Dynamic Range of Regulation: Prokaryotes v. Eukaryotes A. E.coli ON:OFF = :1 max. Most “OFF” genes about 100 x below ON B. Most Eukaryotes ON:OFF = 10 8 :1

24 Thinking About Eukaryotic Txn 2. Genome size How do Regulatory proteins find their targets in the face of 1000 fold increase in “non-specific” DNA? 3. Chromatin and Higher order DNA packaging Concept: Euk genomes are more complex; therefore, the txn machinery is more complex

25 Three Eukaryotic RNAPs RNAP I -> rRNA genes RNAP II -> protein coding RNAP III ->tRNA, 5S rRNA, other small RNAs Basic Machinery Conserved Yeast -> Humans

26 DNA Sequence Elements and DNA Binding Proteins DNA sequence elements that regulate transcription typically bind specific regulatory proteins or protein complexes “cis” sequence elements “trans” factors = proteins or complexes

27 Eukaryotes: Tighter regulation Larger range of regulation Larger genome Multicellular Chromatin More Complex Regulation

28 Enhancers, Activators Promoter, Basal Factors = General Factors Language Caution: Genetic Activator vs. a Txn Activator protein = Activator

29 Watson 9-6 and 9-8 Enhancers= short regions (typically ~ 200 bp) of densely packed consensus elements Some elements found in both promoters and enhancers

30 Lodish Eukaryotic txn = large protein complexes

31 Lodish Complex of complexes ~ 100 proteins

32 Lodish Txn in the face of chromatin and higher order packing

33 Enhancers act independently and cumulatively Reporter Genes

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37 E1 PrCoding RegionE1 PrReporter Cod. Reg. Reporter Genes typically code for easily visualized protiens: lacZ = enzyme: colorless precursor -> blue product GFP = Green flourescent protein (Jellyfish)

38 Reporter Genes

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43 E1PrCoding RegionGFP For Sub-cellular Localization For Expression Pattern (and subcellular local): txn and translation E1PrGFP For Txn Pattern:

44 E1PrMyo2GFP For Expression Pattern: txn and translation

45 Sub-cellular localization of splicing factors Splicing Factor-GFP fusion

46 Ab Protein Expression Pattern Gene Gene (Organism 2 ) Mutant Gene Biochemistr y Genetics Mutant Organism

47 Molecular Genetics Summary 1.Column Chromatograpy (ion exchange, gel filtration) 2.A. Make Polyclonal Ab; B. Make Monoclonal Ab 3.Western blot, in situ immuno-fluorescence (subcellular, tissue) 4.Screen expression library (with an Ab) 5.Screen library with degenerate probe 6.Protein expression (E. coli) 7.A. Differential hybridization 8.A. Northern blot, in situ hybridization, GFP reporter, GFP Fusion 9.A. low stringency hybridization; B. computer search/clone by phone; C. computer search PCR 10.Clone by complementation (yeast, E. coli) 11.A. Genetic screen; B. genetic selection 12.RNAi “knockdown”

48 DNA Sequence Elements and DNA Binding Proteins DNA sequence elements that regulate transcription typically bind specific regulatory proteins or protein complexes “cis” sequence elements “trans” factors = proteins or complexes

49 Regulation: Activation/Repression in Response to Particular Conditions

50 DNA elements (sequence elements) act by binding proteins The proteins do the work

51 Outline Txn in Prokaryotes (Review) Overview of Txn in Eukaryotes Chromatin

52 Fig. 12.1

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54 Fig. 12.5

55 Interphase/prophase Mitosis

56 DNA Compaction: Spaghetti in a Sailboat 2 x 3 x 10 9 bp x.34 nm/bp = 2 meters DNA/nucleus Scale by 1,000,000: Nucleus10  m10 meter sailboat DNA diameter2 nm2 millimeter DNA length2 meters2000 kilometers (1200 miles) DNA persistence length (rigid rod): 50 nm5 cm UIUC to Orlando, Florida = 1066 miles

57 Interphase/prophase Mitosis

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59 Four Core Histones

60 X-Ray Crystallography

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62 Richmond Fig 4 Must Know Sequence of Protein or DNA to Fit to Density Map

63 DNA = Two Turns/Nucleosome

64 Histone Globular Domains and Tails

65 Core Histones are Related Structurally

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68 What are Tails Doing? - Basic = Positively Charged Amino Acids. Tails don’t appear In Crystal Structure = Flexible/Unstructured

69 Tails = Site of Post-Translational Modification

70 Histone Code

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73 Global v. Local Histone Modification

74 Histone H1 = “linker” histone

75 Fig. 12.3

76 Readings Chromatin: Hartwell Heterochromatin: Hartwell

77 Outline Txn in Prokaryotes (Review) Overview of Txn in Eukaryotes Chromatin –Chromosome compaction –Chromatin structure –Heterochromatin and it’s effect on transcription

78 Euchromatin and Heterochromatin Euchromatin = “active” and “decondensed” Constitutive Heterochromatin = always condensed Facultative Heterochromatin = condensed in some cells but not others

79 Barr Body X chromosome inactivation A mechanism of Dosage Compensation

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82 X Chromosome Inactivation Choice of which X is inactivate is random Once inactivated early in development remains inactive throughout cell division and the life of the organism (except in eggs) We can infer two properties: 1. There must be a mechanism(s) of initial inactivation. 2. There must be a mechanism(s) of “duplication” of the inactive state

83 Calico Cats Males = Only Orange or Only Black Females = Orange and Black Mosaics

84 Calico Cats O Gene is on the X chromosome O+ = Black (converts orange pigment to black) o- = Orange Males: XY O+Y = all black, o-Y = all orange (in the regions that show color) Females O+o- = -> black in cells in which o- is inactivated -> orange in cells in which O+ is inactivated

85 Ectodermal Dysplasia X-linked recessive disorder Lack Hair, teeth, sweat glands Mosaic Expression Pattern Clonal = Heritable (Mitosis)

86 The initial inactivation of the X-chromosome and subsequent “maintenance” of inactivation or “duplication” of the inactive state was inferred based on the mosaic nature of the associated phenotypes

87 X Chromosome inactivation is an example of epigenetics Two genes with same promoters and enhancers in the same cell- one is on, the other off. Therefore whether or not a gene is on or off is independent of it’s “normal” genetic regulation. Epigenetic regulation of txn often results from the formation of stable states of chromatin Epigenetic regulation of txn often invovles persitant patterns of histone modification (histone code)

88 X Chromosome inactivation and “non-mosaic phenotypes” Thinking about Hemophelia, diffusable factors and “cells”

89 Outline Txn in Prokaryotes (Review) Overview of Txn in Eukaryotes Chromatin –Chromosome compaction –Chromatin structure –Heterochromatin and it’s effect on transcription X Chromosome inactivation Autosomal heterochromatin and position effect variegation (PEV)

90 Heterochromatin formation and properties Some regions of chromosomes (autosomes) are heterochromatic - genes in these regions are shut off Some regions are euchromatic - genes in these regions are available to be turned on Heterochromatin assembles by a spreading mechanism; assembly starts at a particular site Boundary elements = DNA elements that stop the spreading and define the ends of the heterochromatic regions

91 Heterochromatin Assembly

92 Heterochromatin Duplication Heterochromatin is initially assembled by spreading during development Once it is formed it is copied/duplicated without having to be assembled by spreading de novo each cell division Don’t need boundary elements to keep heterochromatin from spreading: Once a region of heterochromatin is formed it stays the same size through subsequent rounds of mitotic cell division

93 Properties of Heterochromatin Clonal population of cells with the same pattern of heterochromatin: same chromosomal regions inactivated

94 Position Effect Variegation AB B A Chromosomal inversion with one end in a euchromatic region and the other in a heterochromatic region: 1. Moves one of the boundary elements far away 2. changes the order of the genes along the chromosome

95 Y Position Effect Variegation AB BA Heterochromatin initally “spreads” to different extents in different cells in the absence of a boundary element Y

96 Position Effect Variegation BA Heterochromatin initally “spreads” to different extents in different cells in the absence of a boundary element but once formed is “duplicated” during cell division BA BA

97 Y Position Effect Variegation AB BA Heterochromatin initally “spreads” to different extents in different cells in the absence of a boundary element Y Y BA In Cell 1 and its progeny Y is Transcribed: In Cell 2 and its progeny Y is Not Transcribed:

98 Fig a Position effect Variegation

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