Chapter 7: Control of
Gene Expression
Control of Gene Expression
Different cell types differ dramatically in structure and function
►same genome
►Cell differentiation depends on gene expression
Control of Gene Expression
Evidence for preservation of genome during cell differentiation
Control of Gene Expression
Different Cell Types Synthesize Different Sets of Proteins
►How many differences are there btwn any one cell type and another
►Many processes are common to all cells
►Some processes are cell specific
►Cell expresses ~10,000-20,000 of its 30,000 genes; level of expression of almost every
gene varies from cell to cell
Control of gene Expression
Cells Can Change Expression of its Genes in Response to External Signals
Different cell types respond in different ways to same extracellular signal= general
feature of cell specialization
► Example: Liver and adipocyte cells respond differently to glucocorticoid
►
Liver Cell
Tyrosine aminotransferase
Adipocyte
Tyrosine aminotransferase
Control of Gene Expression
For most genes transcription control is most important
Control of Gene Expression
2 Fundamental Components to Transcriptional Gene Regulation
1.
2.
Gene Regulatory Proteins
Short Stretches of DNA of Defined Sequence
Control of Gene Expression
Outside of DNA Helix Read by Proteins
►GRP recognizes specific nucleotide sequence
►Information in form of:
H-bond acceptors
H-bond donors
Hydrophobic patches
►Bind to Major Groove
Control of Gene Expression
GRPs bind to major groove where patterns for ea of four
base-pair arrangements are distinct
Control of Gene Expression
Geometry of Double Helix Depends on Nucleotide Sequence
Some nucleotide sequences cause DNA to bend
►AAAANNN
►If repeated every 10 bp DNA appears unusually curved
Control of Gene Expression
DNA must be flexible for binding of GRPs
Control of Gene Expresion
Short DNA Sequences Fundamental Components of
Genetic Switches
1.
2.
GRP recognition sequence generally < 20 bp
Thousands of such DNA sequences identified ea of which is
recognized by different GRP
Control of Gene Expression
GRP – DNA Interactions
Exact fit btwn DNA and protein
► H-bonds, ionic bonds, hydrophobic
► > 20 contacts
► Tight and specific
►
Control of Gene Expression
Major Structural Motifs of GRPs
1.
2.
3.
4.
5.
Helix-turn-helix
Homeodomain
Zinc Finger
Leucine Zipper
Helix-Loop-Helix
Control of Gene Expression
Helix-Turn-Helix
► Most common
► C-terminal helix= recognition helix
► aa in recognition helix define
specificity
► Structure of GRP varies outside
HTH; HTH presented in unique way
Control of Gene Expression
Homeodomain
►
►
►
►
Special type of helix-turn-helix
Conserved stretch of 60 aa
HTH motif always surrounded by same structurehomeodomain
Master regulators of development
Control of Gene Expression
Zinc Finger Proteins
1.
2.
α helix and β sheet
(2) α helices
Control of Gene Expression
Leucine Zipper
►
►
Clothespin
Helices held together by short
coiled coil region of hydrophobic
residues often leucines
Control of Gene Expression
Helix-Loop-Helix
► Short α helix connected to another via loop
► Flexible loop for packing
Control of Gene Expression
Heterodimerization
►
►
Enhances the repertoire of DNA binding specificities
Combinatorial control
Control of Gene Expression
Is it possible to predict DNA sequence to
which GRP’s bind?
Control of Gene Expression
Gel Mobility Shift Assay to Detect GRPs
►
effect of a bound protein on the migration of DNA in an electric field
Control of Gene Expression
DNA Affinity Chromatography to Purify GRPs
Purification of GRP > 10,000X
Control of Gene Expression
How do we determine the
sequence to which a particular
GRP binds?
Control of Gene Expression
Chromatin Immunoprecipitation
►Identifies sequences occupied by GRPs
in living cells
►Used to identify direct targets of GRPs
How Genetic Switches Work
Tryptophan Operon
Operon= a cluster of genes transcribed as a single mRNA
Operator = short region of DNA in bact. that controls transcription of an
adjacent gene
How Genetic Switches Work
Tryptophan Repressor = a Simple On/Off Switch
How Genetic Switches Work
Repressor= protein binds to DNA to prevent transcription of adjacent gene
Activator = protein that binds to DNA and promotes the transcription of adjacent gene
How Genetic Switches Work
CAP= Catabolite Activator Protein
►Promotes transcription of genes that enable E. coli to use
alternative carbon sources when glucose is not available
►
glucose
cAMP
►cAMP binds to CAP enabling CAP to bind to sequences near
target promoters to promote transcription
How Genetic Switches Work
More complicated genetic switches combine positive and negative controls
Lac Operon- under the control of transcriptional activator
and transcriptional repressor
How Genetic Switches Work
Regulation of Transcription in Eukaryotes is More Complex
1.
2.
3.
GRPs can act even when positioned 1000’s bp away from promoter
RNA Pol II cannot initiate transcription on its own, requires GTFs
Packaging of DNA in chromain
How Genetic Switches Work
Eucaryotic Gene Control Region
►Promoter and all regulatory sequences to which GRPs bind to control transcription
►> 50,000 bp, not unusual
►Packaged in nucleosomes and higher order forms of chromatin
How Genetic Switches Work
Eucaryotic GRPs
►5-10% of human genome
►Vary from one control region to next
►Present in sm amts, <0.01% total protein
►Most recognize specific DNA sequences; others assemble on other DNA bound proteins
►Allow genes to be turned on and off very specifically
How Genetic Switches Work
Eucaryotic Gene Activator Proteins Promote Assembly
of RNA Polymerase and GTFs at Transcription Start
Gene Activator Proteins have Modular Design:
► DNA Binding Domain
► Activator Domain
How Genetic Switches Work
Mechanism of Gene Activator Proteins Varied but All Promote Assembly of GTFs and RNA Pol
►Interact w/ initiation complex to recruit RNA Pol
►Interact directly w/RNA Pol and GTFs
►Change chromatin structure around promoter
How Genetic Switches Work
GRPs can affect:
► prescribed ordered assembly of GTFs and RNA Polymerase
► Recruitment of RNA Polymerase holoenzyme to promoter
How Genetic Switches Work
Gene Activator Proteins Promote Assembly of GTFs and RNA Pol By
►Modification of Local Chromatin Structure Recruiting
histone acetyl transferases
histone remodeling complexes
How Genetic Switches Work
Gene Activator Proteins Work Synergistically
How Genetic Switches Work
EX: Complexity of How Gene Activator Proteins May Ultimately
Increase Transcription Rate
How Genetic Switches Work
Eucaryotic Repressors Inhibit Transcription in Variety of Ways
How Genetic Switches Work
Eucaryotic GRPs and Combinatorial Control
►Function as unit to generate complexes whose
function depends on final assembly of all
components
►Not designated activators or repressors
►DNA acts as nucleation site for assembly
►Can participate in > one type of reg. complex
►Coactivators and corepressors
►enhancesome
How Genetic Switches Work
Eve-skipped gene is a complex multicomponent genetic switch in drosophilia
►Drosophilia development
►Eve expressed when embryo single giant multinucleated cell
►Cytoplasm=mixture of GRPs distributed unevenly along length of embryo
►Nuclei originally identical but later express diff genes cuz exposed to diff GRPs
How Genetic Switches Work
Eve Expression
►Regulatory sequence reads conc of GRPs at ea position along length of embryo
►Expressed in 7 stripes 5-6 nuclei wide precisely positioned along anteriorposterior axis
How Genetic Switches Work
Regulatory Region of Eve Gene
►~20,000 bp binds >20 proteins
►Series of regulatory modules
►Regulatory modules contain multiple reg sequences responsible for
specifying a particular stripe
How Genetic Switches Work
Expression of Stripe 2
►Dictated by 2 gene activator proteins and 2 gene repressor proteins
►Transcription occurs when activators Biocoid and Hunchback are high
and repressors Kruppel and Giant are low
How Genetic Switches Work
Combinatorial Control
Heterodimerization of GRPs in soln
► Assembly of combos of GRPs into sm complexes on DNA
► Many sets of grps bound simultaneous to effect transcription
►
How Genetic Switches Work
Simple regulatory modules= theme of complex
gene regulatory control regions in mammals
5-10% coding capacity of mam genome= GRPs
► Ea gene regulated by set of GRPs
► Ea protein is product of gene that is in turn
regulated by set of other proteins
► Activity of GRPs regulated
►
How Genetic Switches Work
Regulation of Activity of GRPs
How Genetic Switches Work
Human β-globin Gene
►Complex regulation- 2 step process
►Expressed only in RBC at specific time during development
►Possesses own set of GRPs but also under control of LCR
►Cells where no globin gene expressed gene cluster tightly pkged
►Higher order pkging decondensed in RBS
How Genetic Switches Work
LCR= regulatory seq that govern accessibility and expression
of distant genes or gene clusters
►
►
β-thalassemia= deletion in β-globin LCR causing gene to remain
transcriptionally silent–
Many LCRs present in human genome
How Genetic Switches Work
Insulators or Boundary Sequences
►Bind Specialized Proteins
►Regulatory compartmentalization
(Define domains of gene expression)
►Buffer genes from repressing effects of heterochromatin
►Block effect of enhancers (insulator must be btwn enhancer and promoter)
►Mechanism not understood
How Genetic Switches Work
Bacteria use interchangeable sigma subunits to help regulate
transcription while eucaryotes use (3) diff RNA Pol
How Genetic Switches Work
Procaryotes vs Eucaryotes?
Molecular Genetic Mechanisms of
Specialized Cell Types
Cell Memory= prerequisite for the creation of organized
tissues and the maintenance of stably differentiated cell
types
Molecular Genetic Mechanisms of
Specialized Cell Types
Gene Expression and Specialized Cell Types
►
►
►
Environmental effects
Cell memory
Logic circuits
differentiate
keep time
remember events of the past
adjust gene expression over whole chromosome
Molecular Genetic Mechanisms of
Specialized Cell Types
DNA rearrangements mediate phase variation in bacteria
Site Specific Recombination at promoter
Molecular Genetic Mechanisms of
Specialized Cell Types
Rearrangements at the Mat locus determines
mating type in budding yeast
Molecular Genetic Mechanisms of
Specialized Cell Types
Positive Feedback Loops Involving
GRPs can Create Cell Memory
Lambda Repressor and Cro GRPs
Maintain Mode of Growth of
Lambda Phage
Molecular Mechanisms of
Specialized Cell Types
Heritable State of Bacteriophage Lambda
►
►
Switch controls flip-flop btwn lytic and lysogenic state
Governed by two proteins that repress ea other’s
synthesis
 Lambda repressor protein cI
 Cro
►
50 genes in genome
Molecular Mechanisms of
Specialized Cell Types
Lysogenic- bacteriophage DNA integrated into host genome
Lytic- virus multiplies, capsid protein translated and encapsulates
virus which exits host cell and in so doing lysis cell
Molecular Mechanisms of
Specialized Cell Types
Prophage or lysogenic state= lambda repressor occupies operator
synthesis of Cro and
its own synthesis
Lytic State= Cro occupies diff site on operator synthesis of cI and
synthesis its own synthesis to multiply and exit host
Molecular Mechanisms of
Specialized Cell Types
Internal rhythms
►
►
►
►
Governs behavior at diff times of day
Established by day/night cycle
Operates via transcriptional feedback
loop
Resetting clock= destruction of a key
GRP
Molecular Mechanisms of
Specialized Cell Types
Combinatorial control
►Expression of set of genes can be coordinated by single protein
►Effect of single GRP can be decisive
Molecular Mechanisms of
Specialized Cell Types
►Expression of critical GRP can
trigger expression of entire battery
of downstream genes
►Ability to switch many genes on or
off coordinately impt to cell
differentiation
►Conversion of one cell type to
another by single GRP emphasizes
how dramatic differences in cell
types in size, shape, chemistry and
function can be produced by
differences in gene expression
Molecular Mechanisms of
Specialized Cell Types
Combinatorial Gene Control Creates
Many Different Cell Types in Eucaryotes
Molecular Mechanisms of
Specialized Cell Types
Combinatorial Gene Control Creates
Many Different Cell Types in Eucaryotes
Molecular Mechanisms of
Specialized Cell Types
Formation of Entire Organ Coordinated by Single GRP
►Ey coordinates development of Drosophilia eye
Molecular Mechanism of
Specialized Cell Types
Transmitting Stable Patterns of Gene Expression
► Positive feedback loops; GRP activates own expression
► Inhibiting expression an inhibitor to activate and maintain own
expression
► Propagation of chromatin structure
Molecular Mechanism of
Specialized Cell Types
Chromatin states
►heritable
►establish and preserve patterns of gene expression
Molecular Mechanism of
Specialized Cell Types
Mechanisms of Dosage Compensation
► X-inactivation- humans
► Male specific “up-regulation” of transcription- Drosophilia
► Two-fold “down regulation” of X chromosome transcriptionworm
Molecular Mechanism of
Specialized Cell Types
X-inactivation Center
106 nucleotide pairs
Lg regulatory center
Seeds formation of heterochromatin and facilitates its spread
XIST RNA coats inactive chromosome
Molecular Mechanism of
Specialized Cell Types
Role of DNA Methylation in Gene Expression
►
►
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►
Patterns can be inherited
Reinforces transcriptional repression established by other mechanisms
Lock genes in silent state- preventing leaky transcription (106 )
Maintains integrity of genome
Genomic imprinting
Molecular Mechanism of
Specialized Cell Types
Genomic Imprinting
When the expression of a gene is
dependent upon whether it is
maternally or paternally inherited
Molecular Mechanism of
Specialized Cell Types
Maternal: CTCF binds to insulator preventing enhancer from
interacting w/ Igf2 gene= no expression
Paternal: methylation at insulator site prevents CTCF binding
allowing enhancer to interact w/ Igf2 gene = transcription
Molecular Mechanism of
Specialized Cell Types
CG Islands
►Deamination of methylated C’s
nonmutant T
►Deamination of methylated C’s
U which is repaired
►Over evolutionary time 3 out of 4 CGs lost in this way
►Remaining CG unevenly distributed
Posttranscriptional Regulation
Posttranscriptional Controls
►Operate after RNA Pol initiated transcription
►Less common than transcriptional control but
essential in many cases
Posttranscriptional Regulation
Transcriptional Attenuation
►
►
►
Premature termination of transcription
mRNA structure interacts w/ RNA Pol in manner that aborts transcription
Premature termination can be prevented by proteins that bind to mRNA stem
loop
Posttranscriptional Regulation
Alternative Splicing
►Different ways to splice primary transcript resulting in different polypeptides
►Protein complexity can exceed number of genes
►Regulation both positive and negative
Posttranscriptional Regulation
Regulation of RNA cleavage site and Poly-A-addition
►
Changes COOH terminus
► Ex: membrane bound or secreted antibody molecules by B lymphocytes
Posttranscriptional Regulation
RNA Editing
►
►
►
►
►
Posttranscriptional alternation in mRNA sequence
Tranpanosome mitochondrial sequences insertion
of U’s
Plant mitochondrial genes C’s changed to U’s
Mediated by guide RNAs w/ 5’ end complementary to transcript
Mammals deamination of adenine to inosine
which pairs w/ C; mediated by ADARs that
recognize ds RNA structure
Posttranscriptional Regulation
Regulation of nuclear export