GENE REGULATION
Gene regulation is the process of controlling the rate and the manner of gene expression/process that
controls the expression of genes.
In both prokaryotes and eukaryotes, the gene expression is regulated at the transcriptional level. In
eukaryotes it is also regulated at the post-transcriptional level (RNA processing, which take place in the
nucleus, and during translation, which takes place in the cytoplasm and post-translational levels).
. The mechanism is controlled by transcription factors, activators, and repressors. Both prokaryotic and
eukaryotic genes can be regulated to produce multiple gene products.
Gene regulation involves a complex web of interactions within a given cell among signals from the
cell’s environment, signaling molecules within the cell, and the cell’s DNA. These interactions lead to
the expression of some genes and the suppression of others, depending on circumstances.
NOTE: Prokaryotes and eukaryotes share some similarities in their mechanisms to regulate gene
expression; however, gene expression in eukaryotes is more complicated because of the temporal and
spatial separation between the processes of transcription and translation. Thus, although most regulation
of gene expression occurs through transcriptional control in prokaryotes, regulation of gene expression
in eukaryotes occurs at the transcriptional level and post-transcriptional level
GENE REGULATION: OPERON THEORY
Genomic DNA contains both structural genes, which encode products that serve as cellular structures or
enzymes, and regulatory genes, encode products that regulate gene expression. The expression of a gene
is a highly regulated process. Proteins that are needed for a specific function, or that are involved in the
same biochemical pathway, are often encoded together in blocks called operons.
Operons is a group of co-ordinately regulated structural genes with related metabolic functions and the
promoter and operator sites that control transcription. It is also defined as a cluster of genes whose
expression controlled by a single operator. An operon can also be defined as a group of genes physically linked on
the chromosome and under the control of the same promoter(s) (Fig. 6.1). In an operon, the linked genes give rise to a single
mRNA that is translated into the different gene products. This type of mRNA is called a polycistronic mRNA
An operon is essentially consisting of following components.
1. Promoter: A site on the DNA where the RNA polymerase binds and begin the protein
synthesize.
2. Operator: A site on the DNA where the repressor protein binds and blocks the mRNA synthesis
3. Repressor protein: A protein bind in the operator region and block the mRNA synthesis
4. Structural genes: They are the actual coding region of the proteins or enzymes. This protein of
the gene is also referred as open reading frame (orf), because this protein alone is read by RNA
polymerase to transcript the mRNA.
5. Terminator: This portion of DNA of the operan will terminate the mRNA synthesis by forming
loop like arrangement in the same strand.
6. Ribosome binding site: Some 6 base pairs next to promoter, where ribosome bind with mRNA
during translation process.
Each operon includes DNA sequences that influence its own transcription; these are located in a region
called the regulatory region. The regulatory region includes the promoter and the region surrounding the
promoter, to which transcription factors, proteins encoded by regulatory genes, can bind. Transcription
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factors influence the binding of RNA polymerase to the promoter and allow its progression to transcribe
structural genes.
Prokaryotic Gene Regulation
In prokaryotes, there are examples of operons whose gene products are required rather consistently and
whose expression, therefore, is unregulated. Such operons are constitutively expressed, meaning they
are transcribed and translated continuously to provide the cell with constant intermediate levels of the
protein products. Such genes encode enzymes involved in housekeeping functions required for cellular
maintenance, including DNA replication, repair, and expression, as well as enzymes involved in core
metabolism.
In contrast, there are other prokaryotic operons that are expressed only when needed and are regulated
by repressors, activators, and inducers.
In prokaryotic cells, there are three types of regulatory molecules that can affect the expression
of operons: repressors, activators, and inducers. Repressors and activators are proteins produced in the
cell. Both repressors and activators regulate gene expression by binding to specific DNA sites adjacent
to the genes they control. In general, activators bind to the promoter site, while repressors bind to
operator regions.
A repressor is a transcription factor that suppresses transcription of a gene in response to an external
stimulus by binding to a DNA sequence within the regulatory region called the operator, which is
located between the RNA polymerase binding site of the promoter and the transcriptional start site of the
first structural gene. Repressor binding to operator blocks RNA polymerase from transcribing structural
genes.
Equally, an activator is a transcription factor that increases the transcription of a gene in response to an
external stimulus by facilitating RNA polymerase binding to the promoter.
An inducer, a third type of regulatory molecule, is a small molecule that either activates or represses
transcription by interacting with a repressor or an activator depending on the needs of the cell and the
availability of substrate.
In bacteria and archaea, structural proteins with related functions are usually encoded together
within the genome in a block called an operon and are transcribed together under the control of a single
promoter, resulting in the formation of a polycistronic transcript (see Fig. 6.1).
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Fig 6.1: In prokaryotes, structural genes of related function are often organized together on the genome
and transcribed together under the control of a single promoter. The operon’s regulatory region
includes both the promoter and the operator. If a repressor binds to the operator, then the structural
genes will not be transcribed. Alternatively, activators may bind to the regulatory region, enhancing
transcription.
In this manner, regulation of the transcription of all of the structural genes encoding the enzymes that
catalyze the many steps in a single biohemical pathway can be controlled simultaneously, because they
will either all be needed at the same time, or none will be needed e.g. in E. coli, all of the structural
genes that encode enzymes needed to use lactose as an energy source lie next to each other in the lactose
(or lac) operon under the control of a single promoter, the lac promoter.
The lac Operon: An Inducer Operon
It`s is an example of Inducible Transcription. The lac operon in the bacterium E. coli has more complex
regulation, involving both a repressor and an activator. Three genes of the lac operon that encode for
enzyme that are needed to break down/metabolize lactose.
For the lac operon to be activated, two conditions must be met. First, the level of glucose must
be very low or non-existent. Second, lactose must be present. Only when glucose is absent and lactose is
present will the lac operon be transcribed. In the absence of glucose, the binding of the CAP protein
makes transcription of the lac operon more effective.
When lactose is not present, the proteins to digest lactose are not needed/ are usually present in very low
concentrations. Therefore, a repressor binds to the operator and prevents RNA polymerase from
transcribing the operon. Hence transcription is inhibited by a repressor protein produced by a regulator
gene (see the top portion of the Fig.Fig. 6.2).
However, when lactose is present, lactose binds to the repressor and inactivates it, effectively removing
the blockade/removes it from the operator and enabling transcription of the messenger RNA needed for
synthesis of these genes (lower portion of Fig. 6.2). RNA polymerase is now free to transcribe the genes
necessary to digest lactose. The promoter is the site on DNA where RNA polymerase binds in order to
initiate transcription.
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Fig. 6.2. Regulation of the lac operon. Transcription of the lac operon only occurs when lactose is present. Lactose binds to
the repressor and removes it from the operator. Transcription of the lac operon is carefully regulated so that its expression
only occurs when glucose is limited and lactose is present to serve as an alternative fuel source. Lac Z, Lac Y and Lac A
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are the structural genes which code the enzymes namely, Betagalactosidase, lactose permease and
lactose transactylase (which are essential for lactose utilization); T denotes the terminater which stops
the mRNA synthesis. lacZ, lacY, and lacA are coordinately expressed from one promoter called lacP that directs
expression of a polycistronic mRNA
The trp Operon: A Repressor Operon
It`s is an example of Repressible Transcription. E. coli need the amino acid tryptophan, and the DNA in
E. coli also has genes for synthesizing it.
The trp operon includes three important regions: the coding region, the trp operator and the trp
promoter. The coding region includes the genes for the five tryptophan biosynthesis enzymes. Just
before the coding region is the transcriptional start site. The promoter sequence, to which RNA
polymerase binds to initiate transcription, is before or “upstream.
When E. coli needs to synthesize tryptophan, it must express a set of five proteins that are
encoded by five genes. These five genes are located next to each other in the tryptophan (trp) operon.
These genes generally transcribe continuously since the bacterium needs tryptophan.
A DNA sequence called the operator is located between the promoter and the first trp gene. The
operator contains the DNA code to which the repressor protein can bind. The repressor protein is
regulated by levels of tryptophan in the cell.
When tryptophan concentrations are high/when tryptophan is present in the environment, E. coli does
not need to synthesize it, transcription is repressed (turned off) by binding to a repressor protein and
activating it as illustrated in Fig. 6.2. When tryptophan is present in the cell, two tryptophan molecules
bind to the trp repressor. This causes the repressor to change shape and bind to the trp operator. Binding
of the tryptophan–repressor complex at the operator physically blocks the RNA polymerase from
binding, and transcribing the downstream genes. Thus, when the cell has enough tryptophan, it is
preventing from making more.
However, when tryptophan is not present/low in the cell, the repressor has no tryptophan to bind to it,
the trp operon is turned on so that the genes are transcribed, the proteins are made, and tryptophan can
be synthesized. The repressor is not activated and it does not bind to the operator. Therefore, RNA
polymerase can transcribe the operon and make the enzymes to synthesize tryptophan. Thus, when the
cell does not have enough tryptophan, it synthesizes it.
Because the repressor protein actively binds to the operator to keep the genes turned off, the trp
operon is said to be negatively regulated and the proteins that bind to the operator to silence trp
expression are negative regulators.
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Fig. 6.3: The tryptophan operon. The five genes that are needed to synthesize tryptophan in E. coli are
located next to each other in the trp operon. When tryptophan is plentiful, two tryptophan molecules
bind the repressor protein at the operator sequence. This physically blocks the RNA polymerase from
transcribing the tryptophan genes. When tryptophan is absent, the repressor protein does not bind to the
operator and the genes are transcribed.
NOTE: The main difference between prokaryotic and eukaryotic gene structure is that the prokaryotic
gene structure consists of operons and clusters of several functionally-related genes, whereas the
eukaryotic gene structure does not contain operons.
Gene regulation in Eukaryotes
Unlike prokaryotic cells, eukaryotic cells can regulate gene expression at many different levels.
Gene regulation in eukaryotes may occur when the DNA is uncoiled and loosened from nucleosomes to
bind transcription factors (epigenetic level), when the RNA is transcribed (transcriptional level), when
the RNA is processed and exported to the cytoplasm after it is transcribed (post-transcriptional level),
when the RNA is translated into protein (translational level), or after the protein has been made (post-
translational level).
1. Gene Regulation in eukaryotes at epigenetic level
Epigenetic changes are inheritable changes in gene expression that do not result from changes in the
DNA sequence. Eukaryotic gene expression begins with control of access to the DNA. Transcriptional
access to the DNA can be controlled in two general ways: chromatin remodeling and DNA methylation.
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Chromatin remodeling changes the way that DNA is associated with chromosomal histones.
DNA methylation is associated with developmental changes and gene silencing.
Epigenetic Control: Regulating Access to Genes within the
Chromosome
The DNA in the nucleus is precisely wound, folded, and compacted into chromosomes so that it will fit
into the nucleus and organized so that specific segments can be accessed as needed by a specific cell
type.
Figure 6.4: DNA is folded around histone proteins to create nucleosome complexes. These nucleosomes control
The access of proteins to the underlying DNA.
The DNA molecule itself can also be modified by methylation. DNA methylation occurs within a
specific regions called CpG islands.
Chromatin remodeling alters the chromosomal structure (open or closed) as needed. If a gene is
to be transcribed, the histone proteins and DNA in the chromosomal region encoding that gene are
modified in a way that opens the promoter region to allow RNA polymerase and other proteins, called
transcription factors, to bind and initiate transcription. If a gene is to remain turned off, or silenced, the
histone proteins and DNA have different modifications that signal a closed chromosomal configuration
(Fig. 6.5). In this closed configuration, the RNA polymerase and transcription factors do not have access
to the DNA and transcription cannot occur.
Methylation of DNA and histones causes the nucleosomes to
pack together. Translation factors cannot bind to DNA and
genes hence not expressed
Histone acetylation resulting in loose packaging of nucleosomes
Translation factors bind to DNA and genes hence not expressed
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Fig. 6.5: Nucleosomes can slide along DNA. When nucleosomes are spaced closely together (top), transcription factors
cannot bind and gene expression is turned off. When the nucleosomes are spaced far apart (bottom), the DNA is exposed.
Transcription factors can bind, allowing gene expression to occur. Modifications to the histones and DNA affect nucleosome
spacing.
2. Gene regulation in eukaryotes at transcription level
Like prokaryotic cells, the transcription of genes in eukaryotes requires the action of RNA polymerase
to bind to a DNA sequence upstream of a gene in order to initiate transcription.
Nevertheless, unlike prokaryotic cells, the eukaryotic RNA polymerase requires other proteins, or
transcription factors, to facilitate transcription initiation. RNA polymerase by itself cannot initiate
transcription in eukaryotic cells. There are two types of transcription factors that regulate eukaryotic
transcription: General (or basal) transcription factors and Specific transcription factors
1. General (or basal) transcription factors bind to the core promoter region to assist with the
binding of RNA polymerase.
2. Specific transcription factors bind to various regions outside of the core promoter region and
interact with the proteins at the core promoter to enhance or repress the activity of the
polymerase.
The Promoter and the Transcription Machinery
The promoter region is immediately upstream of the coding sequence. This region can be short (a few
nucleotides in length or quite long (hundreds of nucleotides long]. The longer the promoter, the more
available space for proteins to bind. This also adds more control to the transcription process. The length
of the promoter is gene-specific and can differ dramatically between genes. Consequently, the level of
control of gene expression can also differ quite dramatically between genes.
The promoter binds transcription factors that control the initiation of transcription. Within the
core promoter region, 25 to 35 bases upstream of the transcriptional start site, resides the TATA box.
The TATA box has the consensus sequence of 5’-TATAAA-3’. The TATA box is the binding site for a
protein complex called TFIID, which contains a TATA-binding protein. Binding of TFIID recruits other
transcription factors, including TFIIB, TFIIE, TFIIF, and TFIIH. Some of these transcription factors
help to bind the RNA polymerase to the promoter, and others help to activate the transcription initiation
complex.
In addition to the TATA box, other binding sites are found in some promoters. Examples of these
elements are the CAAT box, with the consensus sequence 5’-CCAAT-3’ and the GC box, with the
consensus sequence 5’-GGGCGG-3’. Specific transcription factors can bind to these promoter-proximal
elements to regulate gene transcription. A given gene may have its own combination of these specific
transcription-factor binding sites. There are hundreds of transcription factors in a cell, each of which
binds specifically to a particular DNA sequence motif. When transcription factors bind to the promoter
just upstream of the encoded gene, it is referred to as a cis-acting element, because it is on the same
chromosome just next to the gene. Transcription factors respond to environmental stimuli that cause the
proteins to find their binding sites and initiate transcription of the gene that is needed.
Role of enhancers and repressors regulating gene expression during transcription in eukaryotes
a) Enhancers and Transcription
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In some eukaryotic genes, there are additional regions referred as enhancers, that help increase or
enhance transcription. These regions, are not necessarily close to the genes they enhance. They can be
located upstream of a gene, within the coding region of the gene, downstream of a gene, or may be
thousands of nucleotides away.
Enhancer regions are binding sequences, or sites, for specific transcription factors. When a
protein transcription factor binds to its enhancer sequence, the shape of the protein changes, allowing it
to interact with proteins at the promotor site. However, since the enhancer region may be distant from
the promoter, the DNA must bend to allow the proteins at the two sites to come into contact (Fig. 6.6).
DNA bending proteins help to bend the DNA and bring the enhancer and promoter regions together
(Fig. 6.5).
Fig 6.6:. Interaction between proteins at the promoter and enhancer sites. An enhancer is a DNA
sequence that promotes transcription. Each enhancer is made up of short DNA sequences called distal
control elements. Activators bound to the distal control elements interact with mediator proteins and
transcription factors. Two different genes may have the same promoter but different distal control
elements, enabling differential gene expression.
This shape change allows for the interaction of the specific activator proteins bound to the enhancers
with the general transcription factors bound to the promoter region and the RNA polymerase.
b) Repressors and Transcription
Repressors turning genes expression off. Similar prokaryotic cells, eukaryotic cells also have
mechanisms to prevent transcription. Transcriptional repressors can bind to promoter or enhancer
regions and block transcription. Like the transcriptional activators, repressors respond to external stimuli
to prevent the binding of activating transcription factors.
3. Gene Regulation at Post-transcriptional level in eukaryotes
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The transcribed RNA must be processed into a mature form before translation begins. This processing
that takes place after an RNA molecule has been transcribed, but before it is translated into a protein, is
referred as post-transcriptional modification.
Post-transcriptional step is regulated to control gene expression in the cell. If the RNA is not
processed, shuttled, or translated, then no protein will be synthesized.
Stages of Post-transcriptional level
RNA Splicing, the First Stage of Post-transcriptional Control
In eukaryotic cells, the RNA transcript often contains regions, called introns, after an RNA molecule has
been transcribed, but prior to its departure from the nucleus to be translated in the cytoplasm, the RNA is
processed and the introns are removed by splicing prior to translation (Fig. 6.7). Splicing is done by
spliceosomes, ribonucleoprotein complexes that recognizes two ends of the intron, cut the transcript at
those two points, and bring the exons together for ligation. Exons regions of RNA code for a protein.
Fig. 6.7: RNA Splicing. Pre-mRNA can be alternatively spliced to create different proteins.
Importance of RNA stability in gene regulation /Control of RNA Stability at Post-transcriptional
level in eukaryotes
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Before the mRNA leaves the nucleus, it is given two protective "caps" that prevent the ends of the strand
from degrading during its journey. 5' and 3' exonucleases can degrade unprotected RNAs. The 5' cap,
which is placed on the 5' end of the mRNA, is usually composed of a methylated guanosine triphosphate
molecule (GTP). The GTP is placed "backward" on the 5' end of the mRNA, so that the 5' carbons of the
GTP and the terminal nucleotide are linked through three phosphates. The poly-A tail, which is attached
to the 3' end, is usually composed of a long chain of adenine nucleotides. These changes protect the two
ends of the RNA from exonuclease attack.
After the RNA is transported to the cytoplasm, the length of time that the RNA resides there can
be controlled. Each RNA molecule has a defined lifespan and decays at a specific rate. This rate of
decay can influence how much protein is in the cell. If the decay rate is increased, the RNA will not
exist in the cytoplasm as long, shortening the time available for translation of the mRNA to occur. On
the other hand, if the rate of decay is decreased, mRNA molecule will reside in the cytoplasm longer and
more protein can be translated. This rate of decay is referred to as the RNA stability. If the RNA is
stable, it will be detected for longer periods of time in the cytoplasm.
Binding of proteins to the RNA can also influence its stability. Proteins called RNA-binding
proteins, or RBPs, can bind to the regions of the mRNA just upstream or downstream of the protein-
coding region. These regions in the RNA that are not translated into protein are called the untranslated
regions, or UTRs. They are not introns (those that have been removed in the nucleus). Rather, these are
regions that regulate mRNA localization, stability, and protein translation. The region just before the
protein-coding region is called the 5' UTR, whereas the region after the coding region is called the 3'
UTR
The binding of RBPs to these regions can increase or decrease the stability of an RNA molecule,
depending on the specific RBP that binds.
.
Fig.6.8: RNA-binding proteins. The protein-coding region of this processed mRNA is flanked by 5' and
3' untranslated regions (UTRs). The presence of RNA-binding proteins at the 5' or 3' UTR influences the
stability of the RNA molecule.
RNA Stability and microRNAs
In addition to RBPs that bind to and control (increase or decrease) RNA stability, other elements called
microRNAs can bind to the RNA molecule. The microRNAs, or miRNAs, are short RNA molecules that
are only 21 to 24 nucleotides in length. The miRNAs are made in the nucleus as longer pre-miRNAs.
These pre-miRNAs are chopped into mature miRNAs by a protein called Dicer. Like transcription
factors and RBPs, mature miRNAs recognize a specific sequence and bind to the RNA; however,
miRNAs also associate with a ribonucleoprotein complex called the RNA-induced silencing complex
(RISC). The RNA component of the RISC base-pairs with complementary sequences on an mRNA and
either hinder translation of the message or lead to the degradation of the mRNA.
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4. Gene Regulation at Translational and Post-translational level in Eukaryotic cells
After RNA has been transported to the cytoplasm, it is translated into protein. Control of this process is
largely dependent on the RNA molecule.
The Initiation Complex and Translation Rate
Like transcription, translation is controlled by proteins that bind and initiate the process. In translation,
the complex that assembles to start the process is referred to as the translation initiation complex.
In eukaryotes, translation is initiated by binding the initiating met-tRNAi to the 40S ribosome. This
tRNA is brought to the 40S ribosome by a protein initiation factor, eukaryotic initiation factor-2 (eIF-
2). The eIF-2 protein binds to the high-energy molecule guanosine triphosphate (GTP). The tRNA-
eIF2-GTP complex then binds to the 40S ribosome.
A second complex forms on the mRNA. Several different initiation factors recognize the 5' cap
of the mRNA and proteins bound to the poly-A tail of the same mRNA, forming the mRNA into a loop.
The cap-binding protein eIF4F brings the mRNA complex together with the 40S ribosome complex.
The ribosome then probes along the mRNA until it finds a start codon AUG. When the anticodon
of the initiator tRNA and the start codon are aligned, the GTP is hydrolyzed, the initiation factors are
released, and the large 60S ribosomal subunit binds to form the translation complex. The binding of eIF-
2 to the RNA is controlled by phosphorylation. If eIF-2 is phosphorylated, it undergoes a conformational
change and cannot bind to GTP. Therefore, the initiation complex cannot form properly and translation
is hindered. When eIF-2 remains unphosphorylated, the initiation complex can form normally and
translation can proceed.
Fig.6.9: Gene expression can be controlled by factors that bind the translation initiation complex.
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