5. Regulation of gene expression- in prokaryotes
Molecular biology
Mgr. Marie Brázdová, Ph.D
brazdovam@pharm.muni.cz
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Gene expression - the formation
of proteins or RNA products
Usually, only a small portion of the
genes present in a cell are
expressed at a given time
Gene expression is regulated
differently in prokaryotes and
eukaryotes
Level of regulation:
• transcription
• mRNA stability
• translation
• post-translation
modification
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Gene expression is regulated
differently in prokaryotes and
eukaryotes
In prokaryotic cells, the control of gene
expression is mostly at the transcriptional
level.
• Prokaryotic organisms are single-celled
organisms that lack a cell nucleus, and their
DNA therefore floats freely in the cell
cytoplasm.
• To synthesize a protein, the processes of
transcription and translation occur almost
simultaneously. When the resulting protein is no
longer needed, transcription stops. As a result,
the primary method to control what type of
protein and how much of each protein is
expressed in a prokaryotic cell is the regulation
of DNA transcription.
• All of the subsequent steps occur automatically.
When more protein is required, more
transcription occurs. Therefore, in prokaryotic
cells, the control of gene expression is mostly
at the transcriptional level.
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provide plastic metabolism:
prepare maximum growth and
reproduction under a variety
of conditions
4
The only circular DNA in a cell
DNA is not complexed with
histones
The nucleus is not separated from
the cytoplasm
Gene transcripts do not contain
introns
Translation and transcription take
place simultaneously
Basic features of gene expression in prokaryotes
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 311.
Simpler regulatory mechanisms than
eukaryotes
Regulation takes place at the level of
transcription initiation
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mechanisms by which gene expression is rapidly turned
on or off in response to changes in the environment
provide metabolism plasticity: achieving maximum growth
and reproduction under a variety of conditions
pre-programmed cascades of gene expression
the stimulus affects the expression of a certain gene, the
product of which affects the expression of a set of other
genes, their products another, etc.
1. Regulation of transcription in prokaryotes:
-the only type of RNA polymerase
-yet turning genes on and off as needed
-rapidly changing environmental conditions
-signals to start and stop transcription in the form of small molecule
substrates
-mediating signal transduction to the promoter by the protein
2. Transcription attenuation
3. Riboswitches - altered secondary structure of mRNA
4. Alternative sigma factors
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Regulation gene expression in prokaryotes
Constitutive expression
stable (continuous) expression of genes that encode cell components
necessary for the maintenance of normal-operational-cellular functions
Eg. expression of genes for rRNA, tRNA, ribosome proteins, RNA
polymerases, proteins involved in proteosynthesis, enzymes catalyzing
operational functions
constitutive genes are thus expressed in most cells
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Constitutive, inducible and repressible gene
expression
Inducible / repressible expression
gene expression is increased or decreased as needed
refers to (inducible / repressible) genes whose products are only needed
under certain conditions
the synthesis of these genes is under the control of special regulatory
systems
constitutive expression of these genes would mean an unnecessary energy
load on the cell (evolutionary advantage)
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Constitutive, inducible and repressible gene
expression
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Use of different energy sources in bacteria
several hydrocarbons are usable (eg glucose, sucrose, galactose, arabinose, lactose)
-if available, E. coli prefers the use of glucose
- in the presence of lactose and in the absence of glucose, E. coli cells adapt rapidly: initiates the
synthesis of two enzymes: β-galactosidase and β-galactoside permease
β-galactoside permease transports lactose into the cell
β-galactosidase breaks down lactose into glucose and galactose
in the absence of lactose in the environment, E. coli cells do not synthesize these enzymes
Induction of genes for the use of lactose
• expression of both genes is rapidly induced in
the absence of glucose and in the presence of
lactose
• The process of turning on gene expression by a
substance delivered to the environment in
which a cell grows is called induction
• the genes whose expression is thus regulated
are called inducible
• induction occurs at the level of transcription
and changes the number of molecules of the
respective proteins (not the activity of already
existing proteins)
• typical inducible enzymes are enzymes involved
in catabolic (degradation) pathways
Repression of genes for tryptophan synthesis
• bacteria can synthesize most of the organic molecules necessary for their growth (amino
acids, purines, pyrimidines, etc.)
• eg in the E. coli genome there are 5 genes encoding enzymes involved in tryptophan
biosynthesis
• their expression is necessary in an environment lacking tryptophan (to ensure
proteosynthesis)
• in the environment with tryptophan the expression of these genes is unnecessary,
regulatory mechanisms ensure their repression (attenuation)
• in the absence of tryptophan, derepression occurs (expression of the relevant genes is
switched on)
• repression occurs at the
transcriptional level
• enzymes involved in anabolic
(synthetic) processes are
often repressible
• repression is not the same as
negative feedback (the
product of the biosynthetic
pathway inhibits the activity
of the first enzyme of the
pathway)
Repression of genes for tryptophan synthesis
Summary TEST
• in prokaryotes, genes providing operational functions (rRNA, tRNA,
ribosome proteins) are constitutively expressed; other genes are
usually expressed depending on the need for their products
• genes that encode enzymes of catabolic pathways are often
expressed inducibly; only in the presence of appropriate substrates
• the expression of genes encoding enzymes of anabolic (synthetic)
pathways is usually switched off in the presence of the end
product of this pathway; they are repressible
• gene expression can be regulated at many levels, but regulation of
transcription is most common
Positive and negative regulation
of gene expression
• the cell has regulatory proteins that can induce or inhibit the
expression of one or more genes
• they are encoded by regulatory genes
• positive regulatory mechanisms turn on the expression of
structural genes
• negative regulatory mechanisms turn off the expression of
structural genes
• in both cases inducible and repressible systems can be
applied
• regulatory proteins bind to DNA at a
regulator protein binding site (RPBS)
adjacent to the structural gene
promoter
• in positive regulatory systems, regulatory
proteins are termed activators because,
upon binding to RPBS, they activate the
transcription of structural genes
• in negative regulatory systems, regulatory
proteins are called repressors because,
after binding to RPBS, they repress the
transcription of structural genes
Activators and repressors Effector molecules
• the binding of a regulatory protein
to the RPBS site depends on the
presence of effector molecules
• effector molecules are usually
small molecules (amino acids,
sugars, etc.)
• effector molecules involved in the
induction of gene expression are
termed inducers
• effector molecules involved in the
repression of gene expression are
termed corepressors
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Basic concepts
• repressor is a protein encoded by regulatory genes that bind to DNA to stop
transcription of an operon.
• operator is a DNA sequence outside the operon to which a specific repressor
protein regulating the functionality of a structural gene binds.
• operon is a unit that coordinates and regulates gene activity in prokaryotes controlling
protein synthesis. It contains regulatory elements and genes encoding
proteins.
• inducer is a compound that leads to the production of a larger amount of a given
protein (enzyme) in a cell, by direct binding to the regulatory sequences of its
gene.
• promoter is the 5´-non-coding sequence of the gene to which RNA polymerase
binds.
• regulator: any substance that is involved in the regulation of molecular processes
• regulatory protein: the self-entity involved in regulation usually binds to the
promoter
• allosteric effector: compound that leads to the production of conformation
changes of protein
• Activator protein that binds to prokaryotic operators to increase transcription
• catabolite activator protein (CAP) protein that complexes with cAMP to bind to the promoter
sequences of operons which control sugar processing when glucose is not available
• inducible operon operon that can be activated or repressed depending on cellular needs and the
surrounding environment
• lac operon - operon in prokaryotic cells that encodes genes required for processing and intake of
lactose•
negative regulator- protein that prevents transcription
• Operator- region of DNA outside of the promoter region that binds activators or repressors that
control gene expression in prokaryotic cells
• Operon- collection of genes involved in a pathway that are transcribed together as a single mRNA in
prokaryotic cells
• positive regulator- protein that increases transcription
• Repressor- protein that binds to the operator of prokaryotic genes to prevent transcription
• transcriptional start site- site at which transcription begins
• trp operon- series of genes necessary to synthesize tryptophan in prokaryotic cells
• Tryptophan- amino acid that can be synthesized by prokaryotic cells when necessary
• -
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Participants in gene expression
regulation:
regulator:
any substance that is involved in the
regulation of molecular processes
regulatory protein
own entity involved in regulation,
they mostly bind to the promoter
allosteric effector
low-molecular substance, which
changes its binding to the regulatory
protein and its affinity for the
regulated region
Types of regulators:
positive regulator
induces transcription, translation
negative regulator
stops transcription, translation
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Activation by recruitment
of RNA polymerase. (a) In the
absence
of both activator and repressor, RNA
polymerase occasionally binds the
promoter
spontaneously and initiates a
lowlevel (basal
level) of transcription. (b) Binding of
the repressor
to the operator sequence blocks
binding of RNA polymerase and so
inhibits
transcription. (c) Recruitment of RNA
polymerase
by the activator gives high levels of
transcription. RNA polymerase is
shown recruited
in the closed complex (see Fig.
13-3). It then spontaneously
isomerizes to
the open complex and initiates
transcription.
If both the repressor and activator
are
present and functional, the action of
the repressor
typically overcomes that of the
activator.
(This case is not shown in the figure.)
Allosteric effect:
interaction of the effector with the regulatory protein
RP
E
RP
RO
change in conformation of a regulatory protein
creation or destruction of a binding site for the
regulatory region
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➢change in the
conformation of
the regulatory
protein
➢destruction or
creation of a
binding site for
the regulatory
region
➢interaction of the
effector with the
regulatory protein
Types of allosteric effectors:
negative allosteric effector
prevents binding of the
regulatory protein to the
regulatory region
positive allosteric effector
allows binding of the regulatory
protein to the regulatory region
http://teachmephysiology.com/basics/enzyme-activity/enzyme-inhibition/
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Types of regulatory proteins:
negative
their binding to the regulatory region prevents RNA polymerase from transcribing the transcription
unit
positive
their binding to the regulatory region allows RNA polymerase to transcribe the transcription unit
transcription activators
➢Síť regulačních vztahů:
regulátor
negativnípozitivní
zastavuje
transkripci
navozuje
transkripci
molekulární
efektory
regulační
proteiny
pozitivní negativní pozitivní negativní
vážou se na
regulační
proteiny
vážou se na
regulační
proteiny
vážou se na
regulační
oblast
navozuje
transkripci
zastavuje
transkripci
umožňuje vazbu
regulačního proteinu
na regulační oblast
zabraňuje vazbě
regulačního proteinu
na regulační oblast5_regulace_2020FaF 20
21
RNA polymerase regulation by
repressor - negative control
repressor is encoded by a regulatory gene
After synthesis, the repressor diffuses to the promoter and binds in a region called the
operator (usually part of the promoter)
The repressor blocks RNA polymerase binding to the promoter
mRNA synthesis does not occur
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 312.
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regulatory gene
REPRESOR
22
The repressor is controlled by two mechanisms
Induction Corepression
inductor is a small
molecule that binds
to a repressor,
changes its
conformation, and
causes
disconnection from
the operator
Transcription can
begin
Inducers: small
molecules of
nutrients or their
metabolites
repressor is not
active unless the
corepressor is
attached to it. The
repressorcorepressor
complex
binds to DNA and
prevents RNA
polymerase binding
There is no
transcription
Corepressors: small
molecules of
nutrients or their
metabolites
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Inducible systems
Repressible systems
25
Operon theory
Structural genes in
bacteria are often
grouped into operons
NOVÁK, Jan. Biochemie I. Brno: Muni, 2009, s. 311.
Operon
•Includes structural genes for metabolically coupled proteins
• The genes in the operon are usually expressed in a coordinated
way (they are either all "off" or all "on")
• The transcription product is polycistronic mRNA
• Transcription is regulated by a single promoter
•RNA polymerase is regulated - positively or negatively
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Operon
promotor gen A gen B gen C terminátor
Poslední
nukleotid
terminátoru
Startovací
nukleotid
AUG AUG AUG
DNA
RNA – primar transcript
RNA
polymerase
TRANSKRIPCE
• THE PROMOTOR MAY OVERLAPP WITH THE OPERATOR
• The size of the individual regions does not correspond to reality, the genes are in fact much
• larger than the regulatory regions
operator
REPRESOR
gen A gen B gen C terminátor
Operon regulation - positive and negative operon regulation:
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1) Negative regulation of the operon:
is the essence of enzyme induction and
repression
binding of the active repressor to the
operator stops transcription
2) Positive regulation of the operon:
is the essence of catabolic repression
binding of CAP to a promoter in the
presence of an inducer induces
transcription
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Operon regulation - positive and negative operon regulation:
TEST
Inducible operon
•free repressor binds to the
operator and thus sterically
prevents RNA polymerase
from initiating transcription of
structural genes transcription
turned off
• by binding the inducer, the
repressor is inactivated - it
loses the ability to bind to the
operator - transcription is
switched on
➢Enzyme induction (negative regulation):
most often refers to the induction of enzymes induced by inducers
In general, genes, proteins, enzymes…
➢inducible enzymes
➢constitutive enzymes
➢their synthesis does not depend on the presence of an inducer
➢they are formed in the cell in a constant amount
promotor operator geny terminator
represor
inducer-inactivates the repressor
transkripce
inactive complex
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gene transcription takes place, induced enzymes are formed
transkripce genů probíhá - tvoří se indukovatelné enzymy
induktor - inactivation
represor
inaktivní komplex
promotor operator geny terminator
represorrepresor transkripce
induktor
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31
1) Example of induction
Induction of the lac operon by lactose in E.
coli
Enzymes for glucose metabolism by glycolysis
are constitutively produced in E. coli
When lactose is added, the cells adapt and
begin to produce other enzymes encoded by the
lac operon
Allolactose (an isomer of lactose formed
spontaneously) serves as an inducer, binds to
and inactivates the repressor.
RNA polymerase can bind to the promoter and
transcribe structural genes in the lac operon to
produce polycistronic RNA, which encodes three
other enzymes (β-galactosidase, permease, and
transacetylase)
* The process takes place when there is a lack
of glucose in the cell 5_regulace_2020FaF 31
Repressible
operon
•free repressor cannot bind to
operator - transcription
enabled
• the binding of the
corepressor to the repressor
restores the ability of the
repressor to bind to the
operator - transcription
disabled
2) Enzyme repression: corepression
-it most often concerns enzymes of biosynthetic pathways
- the synthesis of these enzymes is suppressed by a specific metabolite
of a given metabolic pathway, which accumulates in a critical amount and
stops further synthesis
- synthesis resumes as soon as the metabolite concentration falls below
a critical value
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Example of corepression
Corepression of trp operon (tryptophan synthesis in E.coli)
The genes for tryptophan synthesis enzymes (5 enzymes in total) are
concentrated in the trp operon
Tryptophan is a corepressor, it binds to an inactive repressor, it changes
its conformation.
The tryptophan-repressor complex inhibits operon transcription.
3.
http://www.mun.ca/biology/scarr/bio4241_chapter13.htm
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Corepression of trp operon (tryptophan synthesis in E.coli)
The genes for tryptophan synthesis enzymes (5 enzymes in total) are concentrated in the trp operon
Tryptophan is a corepressor, it binds to an inactive repressor, it changes its conformation.
The tryptophan-repressor complex inhibits operon transcription.
represor
gene transcription does not take place - corepressor synthesis stops
promotor operator geny terminator
corepressor – activatione
represor
represor
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Lactose operon in E. coli: induction and catabolic
repression
The structural genes of the lac operon are transcribed only in the
presence of lactose and in the absence of glucose
Lac operon induction
• the lac I gene encodes a repressor
• in the absence of an inducer, the repressor binds to the lac
operator and blocks the transcription of structural genes
• the inducer is allolactose, which is formed from lactose by a bgalactosidase
catalysed reaction
• by binding of allolactose to the repressor, the repressor is
released from the operator, the transcription of structural genes
is thus switched on
Reaction catalyzed by
-galaktosidase
5_regulace_2020FaF 39
Structure of the lac operon
• The lac operon contains three genes: lacZ, lacY,
and lacA. These genes are transcribed as a
single mRNA, under control of one promoter.
• Genes in the lac operon specify proteins that
help the cell utilize lactose. lacZ encodes an
enzyme that splits lactose into
monosaccharides (single-unit sugars) that can
be fed into glycolysis. Similarly, lacY encodes a
membrane-embedded transporter that helps
bring lactose into the cell.
• The lacZ gene encodes an enzyme called βgalactosidase,
which is responsible for splitting
lactose (a disaccharide) into readily usable
glucose and galactose (monosaccharides).
• The lacY gene encodes a membrane protein
called lactose permease, which is a
transmembrane "pump" that allows the cell to
import lactose.
• The lacA gene encodes an enzyme known as a
transacetylase that attaches a particular
chemical group to target molecules. It's not
clear if this enzyme actually plays any role in
lactose breakdown.
In addition to the three genes, the lac operon also contains a number of regulatory DNA
sequences. These are regions of DNA to which particular regulatory proteins can bind,
controlling transcription of the operon.
the promoter is the binding site for RNA polymerase, the enzyme that performs
transcription.
The operator is a negative regulatory site bound by the lac repressor protein. The operator
overlaps with the promoter, and when the lac repressor is bound, RNA polymerase cannot
bind to the promoter and start transcription.
The CAP binding site is a positive regulatory site that is bound by catabolite activator
protein (CAP). When CAP is bound to this site, it promotes transcription by helping RNA
polymerase bind to the promoter.
Catabolic repression
• glucose prevents the induction of the lactose
operon: this ensures the preferential use of
glucose instead of less efficient energy sources
• The lac promoter has two components
• RNA polymerase binding site
• catabolite activator protein (CAP) binding site
• binding of the CAP to the promoter activates
transcription of the lac operon
• CAP binds to the promoter only in the presence of
a sufficient level of cyclic AMP (cAMP) - cAMP
functions as an effector molecule
• the level of cAMP is under the control of glucose
(glucose prevents the activation of adenylate
cyclase, ie the enzyme that catalyzes the
formation of cAMP)
Organization of the lac operon in the promoter-operator
CAP positively
regulates the
lac operon,
cAMP is an
effector
• In the presence of glucose
• adenylate cyclase is inactive
• cAMP levels are low
• CAP cannot bind to a lac
operon
• the structural genes of the lac
operon are not expressed
• operator
• In the absence of glucose
• adenylate cyclase is active
• cAMP levels are high
• CAP / cAMP binds to the lac operon
• the structural genes of the lac operon are expresse
5_regulace_2020FaF 42
Catabolite
activator protein
(CAP; also known
as cAMP receptor
protein, CRP) is a
trans-acting
transcriptional
activator that
exists as a
homodimer in
solution.
➢Enzyme induction:
➢2) Catabolic repression:
➢the substrate suppresses the synthesis of inducible enzymes even in the presence of an
inducer
➢Example: Galactose suppresses β-galactosidase synthesis even in the presence of lactose
as an inducer
promotor operator genes terminator
represorrepresor
induktor inactivate represor
RNA polymerase has a low affinity for the promoter - it does not bind and
transcription occurs at a low frequency
promotor operator genes terminator
CAP-cAMP- complex increases the affinity of RNA polymerase for the promoter
CAPCAP
transcription
represorrepresor inactive
affinity of RNA polymerase for promoter-binding transcription is ongoing
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5_regulace_2020FaF 44
➢Regulation- lac operonu:
https://www.slideshare.net/alokbharti18/regulation-of-gene-expression
5_regulace_2020FaF 45
1.
2.
5_regulace_2020FaF 46
Attenuation
• Attenuation means the premature
termination (interruption) of
transcription by a change in the
secondary structure of the mRNA. It
occurs when a cell does not need a
transcription product. Part of the
operon is an area called the
attenuator.
• In prokaryotes, both transcription
and translation take place at the
same time. Imagine that behind the
RNA polymerase, which produces
mRNA for tryptophan synthesis
proteins, the ribosome is already
moving and forming these proteins.
As RNA polymerase approaches the 5
'end of the transcript, many codons
for tryptophan appear.
• An example is the trp operon that
produces tryptophan:
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http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-23/23_08.jpg5_regulace_2020FaF 48
Riboswitch:
secondary structure of mRNA capable of binding any ligand
Binding of the ligand to the mRNA changes its secondary
structure - "on" or "off" gene expression
http://www.umich.edu/~rnapeopl/WalterSummaryRiboswitch.htm
5_regulace_2020FaF 49
riboswitch is a regulatory segment of a
messenger RNA molecule that binds a
small molecule, resulting in a change in
production of the proteins encoded by
the mRNA.[Thus, an mRNA that contains
a riboswitch is directly involved in
regulating its own activity, in response to
the concentrations of its effector
molecule. The discovery that modern
organisms use RNA to bind small
molecules, and discriminate against
closely related analogs, expanded the
known natural capabilities of RNA
beyond its ability to code for proteins,
catalyze reactions, or to bind other RNA
or protein macromolecules.
Transkripce
Translace
TEST
Prokaryotic RNA polymerase
• It recognizes the promoters of all transcription units
• Composed of 5 types of subunits
• 2x α
• 40 kDa - maintains the stability of the complex
• 1x β
• 155 kDa - Key for rNTP binding to enzyme
• 1x β ‘
• 160 kDa - Key for template DNA template binding
• 1x ω
• 160 kDa - regulatory and stabilization roles
• 1x σ
• 85 kDa - the so-called σ-factor - Key for binding to the promoter - several
"interchangeable" variants, each providing binding to a different type of
promoter
• The rate of polymerization is about 15-20 nt / s - depends on the presence of
σ-factor, ribosomes and the like
Alternative sigma factors: Sigma factor (σ factor) is required to initiate
protein transcription in bacteria. It is a
bacterial transcription initiation factor that
allows specific binding of RNA polymerase
(RNAP) to gene promoters.
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