PROKARYOTIC AND

EUKARYOTIC
GENE REGULATION

Jonathan J. Peters, BSc., MSc.

College of Science and Technology
University of University of Liberia
2 Gene Regulation
 Why regulate gene expression?
 Gene expression is expensive, inappropriate gene expression can be
harmful to cells/organisms.
 Thus the proper expression of the phenotype of an organism is
dependent upon expression and lack of expression of genes at
appropriate times and in appropriate cells/places.
 Also, it takes a lot of energy to make RNA and protein.
 Therefore some genes are active all the time because their products
are in constant demand.
 Others are turned off most of the time and are only switched on when
their products are needed.
3 Gene Regulation

 The control of gene expression is much more complex in eukaryotes than in

prokaryotes.

 Reasons being, Eukaryotes have:

 Compartmentalization of cells
 More extensive transcript processing
 Regulation from a distance
 Cell and tissue specific gene expression
 Larger Genome size
 Genes scattered about the genome
4 Gene Regulation in Prokaryotes
 One way in which prokaryotes control gene expression is to group
functionally related genes together so that they can be regulated
together.
 This grouping is called an operon.
 The clustered genes are transcribed together from one promoter giving a
polycistronic messenger.
 An operon can be defined as a cluster gene that encode the proteins
necessary to perform coordinated function.
 Genes of the same operon have related functions within the cell and are
turned on (expressed) and off together (suppressed).
 The first operon discovered was the lac operon so named because its
products are involved in lactose breakdown.
5 Gene Regulation in Prokaryotes

 An operon consists of:

 a promoter (binding site for RNA polymerase)
 a repressor binding site called an operator that overlaps the promoter.
 structural genes
 Operator
 Repressor proteins encoded by repressor genes, are synthesized to regulate gene
expression.
 They bind to the operator site to block transcription by RNA polymerase.
 Promoter
 The promoter sequences are recognized by RNA polymerase.
 When RNA polymerase binds to the promoter, transcription occurs
6 Gene Regulation in Prokaryotes

 Actvators
 The activity of RNA polymerase is also regulated by interaction with accessory
proteins called activators.
 The presence of the activator removes repression and transcription occurs.
 Two major modes of transcriptional regulation function in bacteria (E. coli) to
control the expression of operons:
 repression and
 induction.
 Both mechanisms involve repressor proteins.
 Induction happens in operons that produce gene products needed for the
utilization of energy.
 Repression regulates operons that produce gene products necessary for the
synthesis of small biomolecules such as amino acids.
7
Gene Regulation in Prokaryotes

 Inducible system
 Negative control
 the effector molecule interacts with the repressor protein such that it cannot
bind to the operator
 With inducible systems, the binding of the effector molecule to the repressor:
 greatly reduces the affinity of the repressor for the operator
 the repressor is released and transcription proceeds.
 In addition to negative control mediated by a repressor, expression from an
inducible operon is also under positive control, mediated by an activator.
 A classic example of an inducible (catabolite-mediated) operon is the lac
operon, responsible for obtaining energy from galactosides such as lactose.
8 Gene Regulation in Prokaryotes
 Repressible system
 Negative control
 the effector molecule interacts with the repressor protein such that it
can bind to the operator
 With repressible systems, the binding of the effector molecule to the
repressor:
 greatly increases the affinity of repressor for the operator
 the repressor binds and stops transcription.
 For the trp operon, the addition of tryptophan (the effector molecule)
to the E. coli environment shuts off the system because the repressors
binds at the operator.
 In addition to negative control mediated by a repressor, expression from a repressible
operons is attenuated by sequences within the transcribed RNA.

 A classic example of a repressible operon is the trp operon, responsible

for the biosynthesis of tryptophan.
9 Gene Regulation in Prokaryotes

 Structure of the lac Operon:

 The lac operon contains three structural genes:
Z
y
a
 The z gene codes for β-galactosidase , responsible for the hydrolysis of the
disaccharide, lactose into its monomeric units, galactose and glucose.
 The y gene codes for permease, which increases permeability of the cell to
galactosides.
 The a gene encodes a transacetylase.
 In addition to the structural genes the lac operon also has regulatory genes:
 Promoter: Binding site for RNA polymerase
 Operator: Binding site of repressor
Gene Regulation in Prokaryotes
10
11 Gene Regulation in Prokaryotes

Control of lac operon expression:

The control of the lac operon occurs by both
positive and negative control mechanisms.
Negative control of the lac operon:
What happens to lac operon when glucose is
present and lactose is absent?
During normal growth on a glucose-based
medium (lacking lactose), the lac repressor is
bound to the operator region of the lac
operon, preventing transcription.
12 Gene Regulation in Prokaryotes

 What happens when glucose is absent and lactose is

present?
 The few molecules of lac operon enzymes present will produce a few molecules of
allolactose from lactose.
 Allolactose is the inducer of the lac operon.
 The inducer binds to the repressor causing a conformational shift that causes the
repressor to release the operator.
 With the repressor removed, the RNA polymerase can now bind the promoter and
transcribe the operon
 Positive control: What happens when both glucose and lactose levels are high?
 Since the inducer is present, the lac operon will be transcribed.
 However the rate of transcription is very slow (almost repressed) because glucose
levels are high and therefore cAMP levels are low.
13 Gene Regulation in Prokaryotes
 The repression of the lac operon under these conditions is termed catabolite
repression and is as a result of the low levels of cAMP that results from an
adequate glucose supply.
 This repression is maintained until the glucose supply is exhausted.
 What happens when glucose levels start dropping in the presence of
lactose?
 As the level of glucose in the medium falls, the level of cAMP increases.
 Simultaneously the inducer (allolactose) is also binding to the lac repressor
(since lactose is present).
 The net result is an increase in transcription from the operon.
 The ability of cAMP to activate (increase) expression from the lac operon results
from an interaction of cAMP with a protein termed CRP (for cAMP receptor
protein).
 The protein is also called CAP (for catabolite activator protein).
14 Gene Regulation in Prokaryotes

 The cAMP-CAP complex binds to a region of the

lac operon just upstream of the promoter
 The binding of the cAMP-CRP complex to the lac
operon stimulates RNA polymerase activity 20-to-
50-fold.
 (Repression of the lac operon is relieved in the
presence of glucose if excess cAMP is added.)
 cAMP is therefore an activator of the lac operon.
 This type of regulation by an activator is positive
in contrast to the negative control exerted by
repressors.
15
Gene Regulation in Prokaryotes
16
Gene Regulation in Prokaryotes
 trp operon
 The trp operon encodes the genes for the synthesis of tryptophan.

 As with all operons, the trp operon consists of the promoter, operator and the structural genes.

 It is also subject to negative control by a repressor

 In this system, unlike the lac operon, the gene for the repressor is not adjacent to the promoter, but rather is
located in another part of the E. coli genome.

 Another difference is that the operator resides entirely within the promoter.

 Unlike an inducible system, the repressible operon is usually turned on.

 The operon consists of:

 Structural genes that code for the three enzymes required to convert chorismic acid into tryptophan
 The operon also contains a gene coding for a short oligopeptide (trpL) which functions in attenuation
 Operator
 promoter
17 Gene Regulation in Prokaryotes

Gene Gene Function

P/O Promoter; operator sequence is found in the promoter

trp L Leader sequence; containing attenuator (A)
sequence the leader
trp E Gene for anthranilate synthetase subunit
trp D Gene for anthranilate synthetase subunit
trp C Gene for glycerolphosphate synthetase
trp B Gene for tryptophan synthetase subunit
trp A Gene for tryptophan synthetase subunit
18 Gene Regulation in Prokaryotes

Negative control of trp operon:

 The affinity of the trp repressor for binding the operator region is enhanced when
it binds tryptophan, blocking further transcription of the operon and, as a result,
the synthesis of the three enzymes will decline.
 Hence tryptophan is a co-repressor.
 This means that when tryptophan is absent expression of the trp operon occurs
 The rate of expression of the trp operon is graded in response to the level of
tryptophan in the cell.
19 Gene Regulation in Prokaryotes
20
Gene Regulation in Prokaryotes
21 Gene Regulation in Prokaryotes
22
Gene Regulation in Prokaryotes
23 Gene Regulation in Eukaryotes
 Eukaryotic genes are regulated in units of protein-
coding sequences and adjacent controlling sites, but
unlike prokaryotes, operons are not known to occur.
 Eukaryotic gene regulation is more complex because
eukaryotes possess a nucleus. (transcription and
translation are not coupled).
 Two “categories” of eukaryotic gene regulation exist:
 Short-term - genes are quickly turned on or off in
response to the environment and demands of the
cell.
 Long-term - genes for development and
differentiation.
24 Gene Regulation in Eukaryotes

 Eukaryotic gene expression is regulated at six (6) levels:

1. Transcription
2. RNA processing
3. mRNA transport
4. mRNA translation
5. mRNA degradation
6. Protein degradation
25
Gene Regulation in Eukaryotes
26 Gene Regulation in Eukaryotes

 Transcription control of gene regulation is controlled by:

1. Promoters
• Occur upstream of the transcription start site.
• Some determine where transcription begins (e.g., TATA),
whereas others determine if transcription begins.
• Promoters are activated by specialized transcription factor
(TF) proteins (specific TFs bind specific promoters).
• One or many promoters (each with specific TF proteins)
may occur for any given gene.
• Promoters may be positively or negatively regulated.
27 Gene Regulation in Eukaryotes
2. Enhancers
• Occur upstream or downstream of the transcription start site.
• Regulatory proteins bind specific enhancer sequences; binding is determined by the
DNA sequence.
• Loops may form in DNA bound to TFs and make contact with upstream enhancer
elements.
• Interactions of regulatory proteins determine if transcription is activated or repressed
(positively or negatively regulated).

 More about promoters and enhancers:

• Some regulatory proteins are common in all cell types, others are specific.
• Each promoter and enhancer possesses a specific set of proteins (coactivators) that determines
expression.
• Rate of gene expression is controlled by interaction between positive and negative regulatory
proteins.
• Combinatorial gene regulation; enhancers and promoters bind many of the same regulatory
proteins, implying lots of interaction with fine and coarse levels of control.
28 Gene Regulation in Eukaryotes
 Regulation by histones:
 Eukaryotes possess histones, and histones repress transcription
because they interfere with proteins that bind to DNA.
 If you experimentally add histones and promoter binding
proteins; histones competitively bind to promoters and inhibit
transcription.
 Transcriptionally active genes possess looser chromosome
structures than inactive genes.
 Histones are acetylated and phosphorylated, altering their ability to bind to
DNA.
 Enhancer binding proteins competitively block histones if they are added
experimentally with histones and promoter-binding TFs.
 RNA polymerase and TFs “ step-around ” the histones/nucleosomes and
transcription occurs.
29
Gene Regulation in Eukaryotes

 Short-term - transcriptional control of galactose-utilizing

genes in yeast:
 3 genes (GAL 1, GAL 7, & GAL 10) code enzymes that
function in the galactose metabolic pathway.
• GAL1 galactokinase
• GAL7 galactose transferase
• GAL10 galactose epimerase
 Pathway produces d-glucose 6-phosphate, which enters
the glycolytic pathway and is metabolized by genes that
are continuously transcribed.
 In absence of galactose, GAL genes are not transcribed.
 GAL genes rapidly induced by galactose and absence of
glucose.
 Analagous to E. coli lac operon repression by glucose.
30
Gene Regulation in Eukaryotes

 Hormone regulation - another good example of short-term

regulation of transcription:
 Cells of higher eukaryotes are specialized and generally shielded from rapid
changes in the external environment.
 Hormone signals are one mechanism for regulating transcription in response to
demands of the environment.
 Hormones act as inducers produced by one cell and cause a physiological
response in another cell.
 Hormones act only on target cells with hormone specific receptors, and levels of
hormones are maintained by feedback pathways.
 Hormones deliver signals in two different ways:
• Steroid hormones pass through the cell membrane and bind cytoplasmic
receptors, which together bind directly to DNA and regulate gene expression.
• Polypeptide hormones bind at the cell surface and activate transmembrane
enzymes to produce second messengers (such as cAMP) that activate gene
transcription.
31 Gene Regulation in Eukaryotes
 Hormone regulation continued:
 Genes regulated by steroid hormones possess
binding regions in the sequence called steroid
hormone response elements (HREs).
 HREs often occur in multiple copies in enhancer
sequence regions.
• When steroid is absent:
 receptor is bound and “guarded” by chaperone proteins; transcription does
not occur.
• When steroid is present:
Steroid displaces the chaperone protein, binds the receptor, and binds the
HRE sequence; transcription begins.
32
Gene Regulation in Eukaryotes

2. RNA processing control:

• RNA processing regulates mRNA production from precursor RNAs.
• Two independent regulatory mechanisms occur:
• Alternative polyadenylation = where the polyA tail is added
• Alternative splicing = which exons are splice
 Alternative polyadenylation and splicing can occur together.
 Examples:
• Human calcitonin (CALC) gene in thyroid and neuronal cell
• Sex determination in Drosophilia
3. mRNA transport control:
 Eukaryote mRNA transport is regulated.
 Some experiments show ~1/2 of primary transcripts never leave the nucleus and are
degraded.
 Mature mRNAs exit through the nuclear pores.
33 Gene Regulation in Eukaryotes

4. mRNA translation control:

 Unfertilized eggs are an example, in which mRNAs (stored in the egg/no new
mRNA synthesis) show increased translation after fertilization.
 Stored mRNAs are protected by proteins that inhibit translation.
 Poly(A) tails promote translation.
 Stored mRNAs usually have short poly(A) tails
(15-90 As vs 100-300 As).
 Specific mRNAs are marked for deadenylation (“ tail-chopping ” ) prior to
storage by AU-rich sequences in 3’-UTR.
 Activation occurs when an enzyme recognizes AU-rich element and adds
~150 As to create a full length poly(A) tail.
34
Gene Regulation in Eukaryotes

5. mRNA degradation control:

 All RNAs in the cytoplasm are subject to degradation.
 tRNAs and rRNAs usually are very stable; mRNAs vary
considerably (minutes to months).
 Stability may change in response to regulatory signals and is
thought to be a major regulatory control point.
 Various sequences and processes affect mRNA half-life:
• AU-rich elements
• Secondary structure
• Deadenylation enzymes remove As from poly(A) tail
• 5’ de-capping
• Internal cleavage of mRNA and fragment degradation
35
Gene Regulation in Eukaryotes

6. Post-translational control - protein degradation:

 Proteins can be short-lived (e.g., steroid receptors) or long-lived (e.g., lens
proteins in your eyes).
 Protein degradation in eukaryotes requires a protein co-factor called
ubiquitin. Ubiquitin binds to proteins and identifies them for degradation
by proteolytic enzymes.
 Amino acid at the N-terminus is correlated with protein stability and
determines rate of ubiquitin binding.

• Arg, Lys, Phe, Leu, Trp 1/2 life ≤3 minutes

• Cys, Ala, Ser, Thr, Gly, Val, Pro, Met 1/2 life ≥ 20 hours