Module 4
Regulation of Gene Expression
BIOL 6299
Regulation of gene expression
• All of the different cell
types in a
multicellular organism
have the same DNA
• But control of which
genes are expressed
differs by cell type
• HOW are genes
differentially
expressed?
https://mass.pbslearningmedia.org/resource/tdc02.sci.life.cell.cellgallery/gallery-of-cells/
Ways to control gene expression
Within the nucleus
• Transcription: when and how
different genes are transcribed
• RNA processing: the splicing
and processing of RNA
transcripts
In the cytosol
• RNA transport: which mRNAs
are exported to the cytosol
• mRNA degradation
• Translational regulation:
which mRNAs get translated by
ribosomes
• Protein activation: which
proteins are activated,
inactivated, or degraded
Ways to control gene expression
Within the nucleus
• Transcription: when and how
different genes are transcribed
• RNA processing: the splicing
and processing of RNA
transcripts
• Let’s consider the regulation of
transcription by specific
regulatory protein motif
binding to DNA sequences
In the cytosol
• RNA transport: which mRNAs
are exported to the cytosol
• mRNA degradation
• Translational regulation:
which mRNAs get translated by
ribosomes
• Protein activation: which
proteins are activated,
inactivated, or degraded
DNA major and minor
grooves
Minor groove: has a more acute
angle, is narrow but deep
Major groove: more spacious, is
both wider and deeper
Most proteins interact with DNA at the
major groove (often at consensus
sequences) can form more hydrogen
bonds in the major groove
To control gene expression: consensus sequences
• Consensus sequences are
generally less than 20
nucleotides in length
• Some nucleotides may differ at
different positions
https://i.stack.imgur.com/umTOV.png
Examples of regulatory proteins
that bind DNA at consensus
sequences
• Bacteria
• Lac repressor, CAP, lambda repressor
• Saccharomyces cerevisiae (yeast)
• Gal4, Mata2, Gcn4
• Drosophila melanogaster (the common fruitfly)
• Kruppel, Bicoid
• Mammals
• Sp1, Oct1, GATA1, MyoD, p53
Regulatory protein binding
motifs
• Regulatory proteins control the expression of genes by
interacting with DNA
• These regulatory proteins use common motifs to interact
with DNA in distinct ways:
• Helix-turn-helix (HTH)
• Homeobox domains
• Helix-loop-helix (HLH)
• Leucine zipper
• Zinc finger (ZF)
Helix-turn-helix
motif
• Two alpha helices separated
by a simple bend in the
protein, fits in major groove
• This binding motif was the
first one to be discovered
• Is a very common motif
• Examples of helix-turn-helix
binding: the tryptophan
repressor, CAP fragment
Helix-turn-helix motif interaction with DNA
 Typically, one HTH protein dimerizes
with another HTH protein
 The two sets of
Dimer
formed by
two HTH
proteins
alpha helices,
labeled α2 and
α3, bind DNA
at two
adjacent major
grooves
 Example:
tryptophan
repressor
(more later)
Other DNA binding motifs:
homeobox
• Homeobox domain: a special class of helix-turn-helix
motif
• Homeobox genes are highly conserved, 180 bp DNA
sequence
• Genes encode proteins that act as transcription
factors, 60 amino acid residues in length, with a DNA
binding domain known as the homeo(box)domain
Homeobox
domain
• 3 alpha helices folded
tightly by hydrophobic
interactions
• Helix 3 recognizes the
major groove in DNA
• Helix 1 has a flexible
linker arm that interacts
with the minor groove
in DNA
• Examples: Hox
developmental genes,
antennapedia in fruit
flies
Flexible linker arm
Minor groove
Major groove
DNA
•http://www.biosci.ki.se/groups/tbu/homeo/antp2.gif
Zinc finger motif
• A central zinc atom is bound to
cysteine (C) and histidine (H)
residues
• Chains of varying lengths of
Histidine
amino acids (x=chain length)
extend from the Zn binding
regions
• The zinc finger is part of a larger
protein that binds DNA
• Examples: C2H2 and Gag
knuckle
Zinc (Zn)
in the
center
a.a.
chains
Cysteine
Zinc finger motif
• The zinc chains form
bridges that stabilize alpha
helices and beta-pleated
sheets in the larger protein
• The zinc finger usually
dimerizes and one of the Alpha
alpha helices in the dimer helix
interacts with DNA at a
major groove
• Zinc fingers implicated in
hepatocellular carcinoma
Antiparallel
betapleated
sheets
p53 protein (protruding loop
binding motif)
• p53 protein is a tetramer
• It has an important role as a
tumor suppressor protein
• The protruding loops (L)
recognize and bind DNA at
consensus sites at the major
and minor grooves
p53
Laptenko et al. (2015)
DNA
Leucine zipper (bZIP) binding
motif
• Two alpha helices have
hydrophobic (Leu) regions
and basic (Arg) regions,
form a coiled coil
• The basic regions bind the
major groove of DNA
Leucine
• The hydrophobic regions -rich
are attracted to each
other to form
homodimers or
heterodimers that
resemble a zipper
• Examples: GCN4 in yeast Monomer form
Leucine-rich
Arginine-rich
Dimer form (zipper)
Leucine zipper (bZIP) binding
motif
• Example: GCN4 in yeast
• Leucine zipper binds DNA at the
consensus sequence ATGACTCAT
• The two basic N termini of the
alpha helices fit into major
grooves on opposite sides of the
DNA double helix
• The leucine zipper motif is most
stable when bound to the
consensus sequence
Leucine-rich
Arginine-rich
Dimer form (zipper)
Helix-loop-helix
(HLH) motif
• Two alpha helices separated by loop in
the protein (HLH)
• One alpha helix is generally shorter
than the other and packs tightly against
the other
• Monomers dimerize as homodimers
and bind DNA
• Very common DNA binding motif
important for development, 36 different
genes in animals have this motif
• Aberrations lead to cancer in humans
N-terminus
HLH MONOMER
Helix-loop-helix (HLH) homo and
heterodimers
• Different
combinations of HLH
homodimers and
heterodimers allow
fine-tuning of gene
expression
• Some heterodimers
bind only half as
tightly to DNA and
inhibit transcription
initiation
Torres-Machorro (2021) Int J Mol Sci 23:12855
Example of simple transcriptional
repression
• The tryptophan repressor (helix-turn-helix motif) is an
example of a simple-switch transcriptional repressor that
turns on or off all genes related to synthesis of tryptophan
(tryptophan operon)
• Its regulation is dependent on the availability of amino acids
in the environment
Tryptophan operon in E. coli
5 genes for tryptophan biosynthesis
DNA
mRNA
proteins
 In E. coli, 5 enzymes related to tryptophan biosynthesis are transcribed
under the control of the same promoter region as one long mRNA
 In the presence of tryptophan, transcription of these genes is shut off
Tryptophan gene expression regulation
Tryptophan
 In the absence of
repressor with no
tryptophan or very low
levels, the repressor is
tryptophan present
not bound to the
operator region
 RNA pol binds the
promoter and
transcription of
RNA pol
tryptophan biosynthesis
genes is turned ON
Tryptophan repressor binding
 In the presence of high
tryptophan, tryptophan
binds the repressor,
changing its conformation
 The tryptophan repressor
(a helix-turn-helix motif) is
able to bind DNA at the
operator region
 RNA pol is blocked from
binding at the promoter
region, genes are OFF
Tryptophan repressor
bound by tryptophan
OPERATOR REGION
consensus
sequences
Attenuation of tryptophan
synthesis
• If tryptophan is low but other amino acids needed
for tryptophan synthesis are NOT present, RNA
polymerase will bind but it only has a 10% chance of
remaining in complex with RNA/DNA
• 90% of the time, RNA pol falls off DNA (attenuation)
after only ~162 nucleotides of RNA transcript have
been synthesized
• How does attenuation occur?
Protein expression in bacteria
RNA pol
A
N
D
mRNA
Rib
oso
m
e
Protein
 Remember that in bacteria, transcription and translation take
place simultaneously because DNA is not within a nucleus
Mechanism of tryptophan
attenuation
AUG
mRNA
LEADER sequence
ATTENUATOR
• As RNA pol is transcribing mRNA, the ribosome attaches and
begins translation of mRNA into amino acid residues
• The leader sequence of mRNA gets translated first: Met, Lys, Ala,
Ile, Phe, Val, Leu, Lys, Gly, Trp, Trp, Arg, Thr, Ser STOP
• Translation of the leader sequence requires two tryptophans in a
row
Low tryptophan but other amino acids present
 In the presence
of plenty of
other amino
Ribosome
acids except
tryptophan, the
ribosome stalls
at the two
tryptophans in
a row in the
leader mRNA
 Regions 2 & 3 of mRNA form a stem-loop
sequence
 RNA pol stays bound & continues making mRNA
Attenuation: #3-4 stem loop
forms
Growing protein strand
Ribosome
But in the absence of
other necessary amino
acids, the ribosome stalls
at region 2 and regions 3
and 4 form a stem loop
 Attenuation occurs
 RNA pol falls off DNA
 Transcription ends early
Cross talk to optimize
tryptophan synthesis
• Attenuation allows E. coli to coordinate synthesis of
tryptophan along with the availability of other amino
acids in the cell to optimize production of tryptophan
• Without the other amino acids needed for synthesis,
it is pointless for E. coli to turn on the tryptophan
operon
Phage lambda ƛ repressor: activator
& repressor
• The lambda repressor is another
helix-turn-helix DNA binding
domain
• It operates as both an activator
and a repressor for two different
sets of genes (cI and cro)
• During phage lysogeny, two lambda repressors are bound as dimers to
Operator regions 1 and 2 (OR1, OR2) of phage DNA
• Binding of the lambda repressor helps recruit RNA polymerase to PRM to
begin transcription of cI genes
• Promoter PR is blocked and cro genes are OFFPtashne (2006) Current Biology 16:12:R459-462
Phage lambda ƛ repressor: activator
& repressor
• During the phage lytic
cycle, the lambda
repressor is destroyed
• Without lambda repressor
blocking the operators 1
and 2, RNA polymerase is
able to bind the promoter
PR for cro genes
• cro genes are turned ON
• Lambda phage shifts to transcribing genes essential for lysis of host
• Cro binds operator OR3 to turn off lysogeny genes
Ptashne (2006) Current Biology 16:12:R459-462
More complex gene regulation: E. coli lac operon
 lacZ operon encodes three proteins (lacZ,Y,A) required for the
metabolism of lactose, a disaccharide sugar
 The product of lacZ (beta-galactosidase) breaks lactose into the
monosaccharides glucose and galactose
 Genes are regulated in a coordinated manner via the lac
repressor and CAP proteins
E. coli lac operon
YA
 LacZ (beta-galactosidase) makes a 116 kDa protein that operates as
a tetramer
 LacY encodes permease (lactose transporter)
 LacA encodes thiogalactoside transacetylase
Lac repressor
 Lac repressor is a Vshaped tetramer
 It binds DNA and has
complex control over
gene transcription
 Allows fine-tuning
depending on the
availability of lactose
and glucose
Lac operon repression
RNA
X
CAP Promoter
binding
Operator
site
YA
OFF
 In an environment with no lactose sugar but with glucose present,
the lac repressor is bound to the operator
 RNA polymerase and sigma (σ70) are physically blocked from
binding the promoter region
 lacZ(YA) mRNA is NOT transcribed
Lac repressor binding to
operator
Repression loop formation
 Lac repressor simultaneously binds two different regions of the
operator, bending DNA to form a repression loop
 Equilibrium between repressor binding:
 O1 and O2
 O1 and O3
Lac operon low transcription levels
allo
CAP
lac repressor
ON low
YA
 In an environment with lactose present but with high levels of
glucose present, glucose is preferentially metabolized
 Some allolactose (from lactose) binds the tetrameric lac repressor
and releases the repressor from the operator
 RNA pol binds, and lacZ (at very low levels) is transcribed
Lac operon: high glucose & some lactose present
allo
CAP
lac repressor
ON low
YA
-70 to +1
 Transcription of LacZYA is low because without CAP binding, RNA
pol binds the promoter at a site between -70 and +1
 mRNA is transcribed at low levels beginning at -22 (because this
site is a much less efficient promoter site)
Lac operon: with lactose but low glucose
present
Allolactose binds tetrameric
lac repressor
CAP
ON high
YA
 In the presence of lactose but in the absence of glucose, cyclic AMP
is produced by the cell
 cAMP binds the CAP dimers, and the CAP/cAMP complex binds the
CAP binding site
 CAP enhances the activity of RNA pol so lacZYA transcription is high
Lac operon: with lactose and low glucose
Allolactose binds
tetrameric lac repressor
CAP
ON high
YA
 CAP binding occurs around -52 to -72 bp
 cAMP binding to CAP bends DNA and so RNA pol binds more stably
and efficiently (1000x increase in binding affinity)
Activator proteins bound to
enhancer regions
• Activator proteins
(additional
transcription
factors) are bound
to enhancer
regions which
may be several
kilobases
upstream or
downstream of
the gene they
control
Activator proteins
The activator-mediator complex
• Activators work with
the mediator complex
(25-30 protein
subunits) to bind the
RNA pol II complex
• Allows bending of DNA
to enhance
transcription
• Increased synergy with
multiple activators &
enhancers
Study of role of activators in gene
regulation
Activator protein
bound to enhancer
 Activator proteins may bind DNA 10,000+ nucleotides away from the
promoter region (upstream or downstream)
 Activator proteins help recruit general transcription factors,
mediator, and RNA pol to activate transcription
Role of activators in gene
regulation
 By swapping the
DNA binding
domain and by
adding a
downstream
reporter gene
such as LacZ,
you can study
the necessary
elements of the
DNA recognition
sequences
Structure of chromatin
• Human DNA is long enough to
stretch 300x to the sun and back
• DNA is packaged into tightlywound chromatin inside the
nucleus of a cell
• Chromatin consists of 50%
protein because DNA is wound
around histones (“beads on a
string”)
http://www.nature.com/scitable/topicpage/dna-packaging-nucleosomes-and-chromatin-310
Histone octamers coil DNA into a nucleosome
 Histones are
positivelycharged and bind
well to negativelycharged
phosphates of
DNA
 Nucleosome
formation regulates
transcriptional
access to DNA
https://www.fastbleep.com/biology-notes/40/1191
Methods to alter DNA accessibility
 Replace histones with
other proteins that
allow RNA pol,
mediator and other
general transcription
factors to bind
 Covalently modify
histones to loosen
chromatin and allow
transcription initiation
Methods to alter chromatin
accessibility
 Remodel
nucleosomes with
chromatin
remodeling
complexes to
increase
accessibility
 Remove histones
via chaperone
proteins to loosen
chromatin
Ways that repressors inhibit
transcription
• Compete for binding to the same regulatory sequences on DNA
• Bind the activation domain of an activator protein to block
activation
• Block the assembly of general transcription factors
• Recruit chromatin remodeling complexes to block access to
promoters
• Attract histone deacetylases to turn transcription OFF
• Attract histone methyl transferases to silence genes
• Heterodimerize to inactivate transcriptional activators
Repressors: competitive DNA
binding
 Repressors
bind DNA
and block
binding of
the
activator to
the same
DNA
regulatory
sequence
Repressors: block activation
domain
 Repressors bind the activator to block activation domain
 Without activation, transcription does not proceed
Repressors: interact with general transcription factors
 Repressor
binds general
transcription
factors to
block
activation of
transcription
Repressors: recruit chromatin
remodeling complex
 Repressor
recruits
chromatin
remodeling
complexes to
block access to
transcriptional
activation sites
on chromatin
Repressors: recruit histone
deacetylases
 Repressor
recruits
histone
deacetylases
to turn
transcription
OFF
Repressors: recruit histone methyl
transferases
 Repressor
recruits
histone
methyl
transferase
to silence a
gene or set
of genes
Complexes of regulatory proteins
 Some proteins are
neither activators nor
repressors, but depend
on the assembly of
proteins in a complex
 The entire complex can
repress or activate
transcription
Ways to activate regulatory proteins
Make a new, Ligand-binding Phosphorylate Add a subunit
active protein activation
to activate
the protein
Ways to activate regulatory proteins
Remove an
inhibitor
Allow it to translocate
to the nucleus
Detach it from
the membrane