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Regulation of Gene Expression
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
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
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
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