Regulation of Gene Expression

Dr. Baker
November 28, 2016
 Regulation of Gene Expression: Study Guide
 Reading: Marks, 4th edition, ch 16
 By the end of this unit, the successful student will be able to do the following, as measured by multiple
choice examination:
o Identify the different points where gene expression can be regulated (transcription, processing, etc)
o Define the terms activator, repressor, effector, response elements
o Identify where each is located respective to a eukaryotic gene sequence
o Explain how each would act to regulate gene expression
o Compare and contrast transcriptional regulation in prokaryotes and eukaryotes
o Define ‘operon’
o Describe the lac operon specifically
o Predict the behavior of the lac operon under a given scenario.
o Describe methods of altering gene accessibility
o Compare and contrast chromatin packing and methylation
o Compare and contrast CREB and glucocorticoid gene regulation
o Define RNA processing
o Describe the ways RNA is processed
o Describe non-coding RNAs
o Describe the mechanism behind micro RNA-mediated gene regulation
What is Gene Regulation?
Gene regulation refers to the ability of cells to control
their level of gene expression
Majority of genes are regulated so proteins are
produced at certain times and in specific amounts
This feature is what gives specific cells/tissues their
unique functions
Constitutive genes are unregulated and have
essentially constant levels of expression
 Gene Regulation Can Occur at
Different Points
In both prokaryotes and eukaryotes, most regulation
occurs at the level of transcription
Eukaryotes also have fair amount of regulation at mRNA
processing
Prokaryote Eukaryote
Transcriptional Regulation in Bacteria
Involves regulatory transcription factors
Bind to DNA in the vicinity of a promoter and
affect transcription of one or more nearby
genes
Repressors inhibit transcription
Negative control
Activators increase the rate of transcription
Positive control
Regulation via Transcription Factors
Altering transcription factors is a primary way to mediate
gene expression
Transcription factors may themselves be the regulatory unit
Bind to DNA in the vicinity of the promoter and affect
transcription of one or more genes nearby
Factors that inhibit transcription are called repressors
Factors that increase transcription are called activators
Transcription factors may also be regulated by binding to
small effector molecules
Elements that bind to and regulate how transcription factors can
bind to DNA
Operon
Operon in bacteria is a cluster of genes under
transcriptional control of one promoter
Regulatory region called operator
Transcribed into mRNA as polycistronic mRNA –
encodes more than one protein
Allows regulation of a group of genes with a common
function
Lac operon is an example
 E.coli lac Operon Contains Genes for
Lactose Metabolism  2 regulatory sites
lacO – operator – provides
lacP - promoter
binding site for repressor protein
3 structural genes CAP site – activator protein
lacZ – β-galactosidase binding site
 Allolactose important in lac lacI gene - codes for lac
operon regulation repressor
lacY – lactose permease Considered a regulatory gene
lacA – galactosidase since its sole function is to
regulate other gene’s expression
transacetylase Has its own promoter (not part of
lac operon)
When lactose is absent
Lac repressor protein binds to nucleotides of lac
operator site preventing RNA polymerase from
transcribing lacZ, lacY and lacA
RNA polymerase can bind but not move
forward
When lactose is present
Allolactose is a small effector molecule
4 allolactose molecules binding to lac repressor
prevents repressor from binding
Process called induction (lac operon is
inducible)
Activator Proteins & the Lac Operon
CAP (catabolite activator protein) is
an activator
Example of positive control
Operon is turned off when CAP is not
bound
Small effector molecule, cAMP,
binds to CAP
cAMP binding to CAP induces complex
to bind to CAP site near lac promoter
Resulting bend in DNA enhances RNA
polymerase binding which increases
transcription
Glucose inhibits production of
cAMP and so prevents binding of
CAP to DNA
When both lactose and glucose are high, the lac operon is
shut off
Glucose uptake causes cAMP levels to drop
CAP does not activate transcription
Bacterium uses one sugar at a time, glucose is preferred
When lactose is high and glucose is low, the lac operon is
turned on
Allolactose levels rise and prevent lac repressor from binding to
operator
When lactose is low and glucose is high or low,
the lac operon is shut off
Under low lactose conditions, lac repressor prevents
transcription of lac operon
Eukaryotic Regulation of
Transcription
Follows some of same principles found in
prokaryotes
Activator and repressor proteins influence ability of
RNA polymerase to initiate transcription
Many regulated by small effector molecules
Some important differences
Genes almost always organized individually
Regulation more intricate
‘Combinatorial control’
 How Complicated Can it Get?
1. One or more activator proteins may stimulate the ability
of RNA polymerase to initiate transcription
2. One or more repressor proteins may inhibit the ability of
RNA polymerase to initiate transcription.
3. The function of activators and repressors may be
modulated in several ways, including the binding of small
effector molecules, protein–protein interactions, and
covalent modifications.
4. Activator proteins are necessary to promote the loosening
up of the region in the chromosome where a gene is
located, thereby making it easier for the gene to be
recognized and transcribed by RNA polymerase.
5. DNA methylation usually inhibits transcription, either by
preventing the binding of an activator protein or by
recruiting proteins that cause the DNA to become more
compact.
 Eukaryotic Gene Organization
3 features found in most promoters
TATA box
 25 base pairs upstream from transcriptional start site
 Determines precise starting point for transcription
Transcriptional start site
 With TATA box forms core promoter
 By itself results in low level basal transcription
Regulatory/response elements
 Recognized by regulatory proteins that control transcription initiation
 Activators bind to Enhancers
 Repressors bind to Silencers
Ways Activators/Repressors Regulate

Activators and
repressors
commonly regulate
function of RNA
polymerase II by
binding to general
transcription factors
(GTFs):
Ways Activators/Repressors Regulate
Activators and repressors can also
control RNA polymerase II via
mediator binding
Activators/repressors bind to mediators
located near to the promoter region
Leads to DNA forming a loop structure
Activators stimulate the function of
mediator by allowing faster initiation
Repressors inhibit mediator so RNA
polymerase II cannot progress to
elongation

© 2011 Jones and Bartlett Publishers, LLC (www.jbpub.com)

Ways Activators/Repressors Regulate
Activators and repressors can affect gene accessibility
DNA is associated with proteins to form compact chromatin
Chromatin packing affects gene expression
Transcription is difficult or impossible in the tightly packed
chromatin in the closed conformation
Access to the DNA is allowed in the loosely packed open
conformation
Some activators diminish DNA compaction near a gene
Recruit proteins to loosen DNA compaction
Histone acetyltransferase
ATP-dependent chromatin remodeling enzymes
DNA Methylation as a Means of
Regulation
DNA methylase attaches methyl groups
Common in some eukaryotes but not all
In mammals, 5% of DNA is methylated
CpG islands found near promoter regions
Cytosine and guanine connected by phosphodiester bonds
Unmethylated CpG islands are correlated with active genes
Repressed genes contain methylated CpG islands
Methylation can inhibit transcription in 2 general ways:
Prevent activators from binding to enhancer elements
Converting chromatin from an open to a closed conformation
 Methyl-CpG-binding proteins bind to methylated sequences and recruit
proteins that condense chromatin
 Example of Eukaryotic
Regulatory Circuits
cAMP-dependent activator protein (CREB)
cAMP is a secondary messenger, which can be
produced as a result of several cell-signaling
pathways
cAMP will activate a kinase PKA, which can
translocate from the cytosol to the nucleus
There, activated PKA can phosphorylate (and
activate) CREB
CREB is considered a specific (rather than general)
transcription factor
Upon phosphorylation, it will bind to the promoter
 CREB Pathway

© 2011 Jones and Bartlett Publishers, LLC (www.jbpub.com)

Adapted from B. M. Alberts, et al. Molecular Biology of the Cell, Fifth edition. Garland
 Example of Eukaryotic
Regulatory Circuits
Steroid hormone receptors are
transcription factors that when
bound to hormone activate
transcription.
 Bind to DNA sequences known as
hormone-response elements
within the promoters of responsive
genes
Example: Glucocorticoid
Hormone released into
bloodstream after meals
Transported into cytosol of cells
where it will bind to
glucocorticoid receptors © 2011 Jones and Bartlett Publishers, LLC (www.jbpub.com)
Glucocordicoid binding its
receptor induces the release of
chaperones, thus exposing a
nuclear localization signal
(NLS)
Two glucocorticoid receptors
form a dimer and then travel
through the nuclear pore into
the nucleus
Directed there via NLS
Dimer binds to glucocorticoid
response elements (GREs) that
are next to particular genes
GREs function as enhancer
sequences
Activates the transcription of
the adjacent gene, eventually
leading to the synthesis of the
encoded protein
Regulation via RNA Processing
Unlike bacteria, eukaryotic gene expression is
commonly regulated at the level of RNA
processing and translation
Added benefits include…
Produce more than one mRNA transcript from a
single gene (gene encodes 2 or more polypeptides)
Faster regulation achieved by controlling steps after
RNA transcript made
Modes of regulation include Alternative
Splicing and mRNA Stability
mRNA Stability
mRNA degradation is an important source of
regulating overall gene expression!
While MeG capping and 3’-polyadenylation
have protective effects on the transcript, there
are other factors mediating its stability:
Elements in the 3′ untranslated region (UTR)
determine the stability of an mRNA
The 5′ and 3′ UTRs of the mRNA contain sequences
that regulate translation, stability, cellular
localization of the mRNA
UTR of mRNAs with Short Half-
Lives

Reproduced from an illustration by Rebecca Hartley, University of New Mexico

School of Medicine
 Degradation of mRNA
Occurs primarily by
two pathways, both
begin with the
degradation of the
poly(A) tail, followed
by:
5′ to 3′ exonucleolytic
degradation
or
3′ to 5′ exonucleolytic
degradation by a
complex of proteins
called the exosome
© 2011 Jones and Bartlett
Publishers, LLC
(www.jbpub.com)
Adapted from C. J. Wilusz and J. Wilusz, Trends Genet. 20 (2004):
491-497.
 Non-Coding RNA
ncRNA is RNA that is not tRNA or rRNA, nor does it
code for protein (mRNA)
Long thought to be ‘junk’, they have recently been
found to have important functional roles in gene
regulation
MicroRNA (miRNA) are small RNA molecules that
silence the expression of pre-existing mRNAs
Formerly known as small or short interfering RNA (siRNA)
Contain sequences complementary to specific mRNAs
Are able to knock-down (ie: silence) expression of these
mRNAs
 Current conventions:
 siRNA refers to synthetic double stranded RNAs that are used as laboratory
 Micro RNA (miRNA) Pathway
Primary miRNA
(pri-miRNA) •Dicer’s helicase

DICE
N activity unwinds the
TX

R
70-100 nucleotide
Hairpin structure hairpin to produce a
ssRNA
Drosh
•The RNA-Induced
a 20-25 nt Silencing Complex
Nu
Pre- (RISC) is formed when

DICE
cle miRNA
RISC DICER+ RNA bind

R
us
with Ago proteins

The RISC then cleaves the RNA in

The RISC finds target mRNA the
DICE

via complementary base-pairing region where it is base paired, and

R

RISC
(usually within the 3’UTR) resulting fragments are degraded, thu
silencing gene expression
mRNA -

Complementary sequence
in 3’ UTR
To Review:
Both prokaryotes and eukaryotes regulate gene expression at
the level of transcription, though eukaryotes also have a
large portion of regulation at the level of RNA processing
Transcriptional regulation in prokaryotes can involve
regulatory transcription factors (called activators or
repressors depending on function), and through the binding
of effector molecules to transcription factors to mediate their
function
An operon is a cluster of genes under one promoter. These
genes function together in a single process (the lac operon is
an example of genes working together to metabolize lactose
when in the absence of glucose)
 To Review:
Eukaryotic transcription regulation also may involve regulatory transcription
factors and the binding of effector molecules, but there are also several other
means of regulation
Response elements are mediators (activators or repressors) which bind
upstream of the promoter region, and can affect transcription by causing a
loop-formation in the message which allows the elements to bind to proteins
on or around the promoter
Transcription can also be affected by proteins mediating DNA compaction
(gene accessibility), and methylation of the DNA
Factors affecting RNA processing include alternate splicing of genes, and
pathways affecting stability of the mRNA
Non-coding RNAs (specifically microRNAs) are a newly recognized control
mechanism, wherein RNAs coded for in a genome are complementary to
mRNAs. Binding of these complementary strands activates a degradation
pathway which includes proteins such as dicer and several Ago proteins that
come together and form a complex known as RISC.
Regulation of Gene Expression – Example
Question #1
In liver cells, the presence of glucose will trigger an increase in
cAMP which results in the activation of PKA. What could one
surmise about genes in these cells which contained CREB-
binding elements near their promoter regions?
A. These genes will be silenced in the presence of glucose
B. These genes are most likely involved in the breakdown of
glycogen
C. These genes will only be transcribed when the cell has amassed
enough energy from glycolysis
D. These genes may be involved in the cellular utilization of
glucose
E. Transcription of these genes will be deactivated by PKA
phosphorylation
Regulation of Gene Expression – Example
Question #2
A patient with known lactose intolerance comes to you after ingesting a
quart of sugar-free ice cream, complaining of debilitating stomach cramps
and nausea. Which of the following could you surmise about the
microenvironment of the patients gut at this point?
A. The E.coli in the gut are being exposed to large amounts of lactose but small
amounts of glucose, thus stimulating their lac operons
B. The E.coli in the gut are being exposed to large amounts of lactose and
glucose, thus stimulating their lac operons
C. The E.coli in the gut are being exposed to very low amounts of lactose and
glucose, thus stimulating their lac operons
D. The E.coli in the gut are being exposed to large amounts of glucose but small
amounts of lactose, thus stimulating their lac operons
E. The E.coli in the gut are being exposed to large amounts of lactose but small
amounts of glucose, thus blocking induction of their lac operons