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. Gene Regulatory Proteins
2. 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.

helix and sheet

(2) 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 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 GRPs 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 1000s 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
anterior- posterior 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

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 -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

-thalassemia= deletion in -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 others
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 transcriptionDrosophilia
Two-fold down regulation of X chromosome
transcription- worm

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

Patterns can be inherited

Reinforces transcriptional repression established by other
mechanisms
Lock genes in silent state- preventing leaky transcription (10 6 )
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 Cs

nonmutant T

Deamination of methylated Cs
repaired

U which is

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 Us
Plant mitochondrial genes Cs changed to Us
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