Transcript Reading assignment

MCB 317
Genetics and Genomics
Topic 9
Overview of Eukaryotic Gene Expression
Gene Regulation in
Eukaryotes
Readings
Chromatin: Hartwell Chapter 12, pages 405-410
Heterochromatin: Hartwell Chapter 12, section
12.3
Gene Expression v. Transcription
Concept: Every step in a biological
process is a potential site of regulation
Outline
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•
•
•
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
Expectations and Review
1. Prokaryotes: Basic process and
nomenclature
• Process of txn
• Start and stop signals for txn
• Gene orientation
• One RNAP
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…
Prokaryotes
Consensus sequence
Lodish 11-9
TATA(A/T)A(A/T)(A/G)
Why consensus and not exact sequence?
How Does RNAP “find” its Promoter and Initiate Txn?
Consensus sequences provide for binding to
specific DNA sites over a range of affinities
Concept:
Biological Reactions are often Optimized,
not Maximized
Synthesis/polymerization is in the 5’ to 3’ direction
Coding v. non-coding strand,
Directionality
Coding looks like mRNA
Non-coding can base-pair
With mRNA
Promoters are Directional
Activators
Repressors
Concept:
Turning Genes ON and OFF
ON -> Activated
OFF -> Repressed
OFF -> Not Activated
In General: Repressors Win
Distinguishing: Activators from Repressors
Positive Regulators from Negative Regulators
Key: What is the role of the active form of the protein
Regulation: Activation/Repression in Response to
Particular Conditions
Repressors
Activators and
Repressors vs.
Inducers
Outline
• Txn in Prokaryotes (Review)
• Overview of Txn in Eukaryotes
• Chromatin
Thinking About Prokaryotic v. Eukaryotic
Txn
1. Dynamic Range of Regulation:
Prokaryotes v. Eukaryotes
A. E.coli ON:OFF = 200-1000:1 max.
Most “OFF” genes about 100 x below ON
B. Most Eukaryotes
ON:OFF = 108:1
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
Three Eukaryotic RNAPs
RNAP I -> rRNA genes
RNAP II -> protein coding
RNAP III ->tRNA, 5S rRNA, other small RNAs
Basic Machinery Conserved Yeast -> Humans
DNA Sequence Elements and DNA Binding Proteins
“trans” factors = proteins or complexes
“cis” sequence elements
DNA sequence elements that regulate transcription
typically bind specific regulatory proteins or protein
complexes
Eukaryotes:
Tighter regulation
Larger range of regulation
Larger genome
Multicellular
Chromatin
More Complex
Regulation
Enhancers,
Activators
Promoter,
Basal Factors =
General Factors
Language Caution:
Genetic Activator vs. a
Txn Activator protein
= Activator
Enhancers= short regions (typically ~ 200 bp)
of densely packed consensus elements
Some elements found in both
promoters and enhancers
Watson 9-6 and 9-8
Eukaryotic txn = large protein complexes
Lodish 11-35
Complex of complexes ~ 100 proteins
Lodish 11-36
Txn in the face of chromatin and higher order packing
Lodish 11-37
Enhancers act independently and
cumulatively
Reporter Genes
Reporter Genes
Reporter Genes
E1
E1
Pr
Coding Region
E1
E1
Pr
Reporter Cod. Reg.
Reporter Genes typically code for easily visualized
protiens:
lacZ = enzyme: colorless precursor -> blue product
GFP = Green flourescent protein (Jellyfish)
Reporter Genes
For
Sub-cellular
Localization
For Txn Pattern:
E1
Pr
GFP
For Expression Pattern (and subcellular local): txn and translation
E1
Pr
Coding Region
GFP
For Expression Pattern: txn and translation
E1
Pr
Myo2
GFP
Sub-cellular localization of splicing factors
Splicing Factor-GFP fusion
Biochemistr
y
1
2
Protein
4
Ab
5
6
Expression
Pattern
9
Gene
7
3
Gene (Organism 2)
12
8
10
Mutant Gene
Mutant Organism
11
Genetics
Molecular Genetics Summary
1.
2.
3.
4.
5.
6.
7.
8.
9.
Column Chromatograpy (ion exchange, gel filtration)
A. Make Polyclonal Ab; B. Make Monoclonal Ab
Western blot, in situ immuno-fluorescence (subcellular, tissue)
Screen expression library (with an Ab)
Screen library with degenerate probe
Protein expression (E. coli)
A. Differential hybridization
A. Northern blot, in situ hybridization, GFP reporter, GFP Fusion
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”
DNA Sequence Elements and DNA Binding Proteins
“trans” factors = proteins or complexes
“cis” sequence elements
DNA sequence elements that regulate transcription typically
bind specific regulatory proteins or protein complexes
Regulation: Activation/Repression in Response to
Particular Conditions
DNA elements (sequence elements) act by binding proteins
The proteins do the work
Outline
• Txn in Prokaryotes (Review)
• Overview of Txn in Eukaryotes
• Chromatin
Fig. 12.1
Fig. 12.5
Interphase/prophase
Mitosis
DNA Compaction: Spaghetti in a Sailboat
2 x 3 x 109 bp x .34 nm/bp = 2 meters DNA/nucleus
Scale by 1,000,000:
Nucleus
DNA diameter
DNA length
10m
2 nm
2 meters
10 meter sailboat
2 millimeter
2000 kilometers (1200 miles)
DNA persistence
length (rigid rod): 50 nm 5 cm
UIUC to Orlando, Florida = 1066 miles
Interphase/prophase
Mitosis
Four Core Histones
X-Ray Crystallography
Must Know Sequence of
Protein or DNA to Fit to
Density Map
Richmond Fig 4
DNA = Two Turns/Nucleosome
Histone Globular Domains and Tails
Core Histones are Related Structurally
What are Tails Doing?
- Basic = Positively
Charged Amino Acids.
Tails don’t appear
In Crystal Structure =
Flexible/Unstructured
Tails = Site of
Post-Translational
Modification
Histone Code
Histone Code
Histone Code
Global v. Local
Histone
Modification
Histone H1 = “linker” histone
Fig. 12.3
Readings
Chromatin: Hartwell 465-470
Heterochromatin: Hartwell 479-481
Outline
• Txn in Prokaryotes (Review)
• Overview of Txn in Eukaryotes
• Chromatin
– Chromosome compaction
– Chromatin structure
– Heterochromatin and it’s effect on
transcription
Euchromatin and Heterochromatin
• Euchromatin = “active” and “decondensed”
• Constitutive Heterochromatin = always
condensed
• Facultative Heterochromatin = condensed in
some cells but not others
Barr Body
• X chromosome inactivation
• A mechanism of Dosage Compensation
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
Calico Cats
Males = Only Orange or Only Black
Females = Orange and Black Mosaics
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
Ectodermal Dysplasia
X-linked recessive disorder
Lack Hair, teeth, sweat glands
Mosaic Expression Pattern
Clonal = Heritable (Mitosis)
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
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)
X Chromosome inactivation and “non-mosaic
phenotypes”
Thinking about Hemophelia, diffusable
factors and “cells”
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)
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
Heterochromatin Assembly
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
Properties of Heterochromatin
Clonal population of cells with the same pattern of
heterochromatin: same chromosomal regions
inactivated
Position Effect Variegation
A
B
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
Position Effect Variegation
A
Y
B
Y
B
A
Heterochromatin initally “spreads” to different extents in different
cells in the absence of a boundary element
Position Effect Variegation
B
A
B
A
B
A
Heterochromatin initally “spreads” to different extents in different
cells in the absence of a boundary element but once formed is
“duplicated” during cell division
Position Effect Variegation
A
Y
B
In Cell 1 and its progeny Y is Transcribed:
Y
B
A
In Cell 2 and its progeny Y is Not Transcribed:
Y
B
A
Heterochromatin initally “spreads” to different extents in different
cells in the absence of a boundary element
Position effect Variegation
Fig. 12.14a