Gene Regulation in Eukaryotes

• All cells in an organism contain all the DNA:

– all genetic info
• Must regulate or control which genes are
turned on in which cells
• Genes turned on determine cells’ function
– E.g.) liver cells express genes for liver enzymes but not
Proteins act in trans
DNA sites act only in cis
• Trans acting elements (not DNA) can diffuse through cytoplasm and act at target
DNA sites on any DNA molecule in cell (usually proteins)
• Cis acting elements (DNA sequences) can only influence expression of adjacent genes
on same DNA molecule
Check Points for Gene Expression in Eukaryotes
o Synthesis of proteins is controlled right from the chromatin stage.
o Expression of gene is controlled at many steps during the process of
transcription and translation.
o Description of the control points is dealt in detail in the subsequent slides.
CHROMATIN REMODELLING
Two forms of chromatin
o Euchromatin – A lesser coiled transcriptionally active region which
can be easily accessed by the RNA polymerases.
o Heterochromatin – A highly condensed transcriptionally inactive
region. The genes in this region cannot be accessed by the RNA
polymerases for active transcription.
Mechanisms which affect the chromatin structure and hence the expression of gene
are:
1. Histone modifications – These modifications make a region of gene
either transcriptionally active or inactive.
a) Acetylation
b) ↑Acetylation ----↓ Condensation of DNA ----- ↑ Transcription
of genes in that region
c) Methylation
d) Methylation of histone H4 on R4 (arginine residue at the 4th
position) →→ opens the chromatin structure →→ leading to
transcriptional activation
e) Methylation of histone H3 on K4 and K79 (lysines residues at
the 4th and 79th position) →→ opens the chromatin structure
→→ leading to transcriptional activation
 f) Methylation of histone H3 on K9 and K27 (lysines residues at
the 9th and 27th position) →→ condenses the chromatin
structure →→ leading to transcriptional inactivation
c) Ubiquitination
o Ubiquitination of H2A – Transcriptional inactivation
o Ubiquitination of H2B - Transcriptional activation
2) Methylation of DNA
o Target sites of methylation are - The cytidine residues which exist as
a dinucleotide, CG (written as CpG)↑methylated cytidine --
Transcriptional activity

There are four types of structures of DNA binding proteins,

o Zinc finger proteins
o Helix loop Helix protein
o Leucine zipper proteins
o Homeodomain proteins

Trans-acting factors - factors, usually considered to be proteins, that bind to the cis-
acting sequences to control gene expressionThe protein factors which regulate the
expression of gene by binding to cis acting DNA sequence are termed as trans-acting
elements. Trans-acting molecules generally have two domains: DNA binding domain
(which binds to cis elements) and protein binding domain (required for activation or
suppression of transcription). Transcription initiation is a tightly regulated process
controlled by trans-acting elements both in prokaryotes as well as eukaryotes.
 o

Stages of eukaryotic gene expression (any of which can be potentially regulated).

1. Chromatin structure. Chromatin may be tightly compacted or loose and open.
2. Transcription. An available gene (with sufficiently open chromatin) is transcribed to
make a primary transcript.
3. Processing and export. The primary transcript is processed (spliced, capped, given a
poly-A tail) and shipped out of the nucleus.
4. mRNA stability. In the cytosol, the mRNA may be stable for long periods of time or
may be quickly degraded (broken down).
5. Translation. The mRNA may be translated more or less readily/frequently by ribosomes
to make a polypeptide.
6. Protein processing. The polypeptide may undergo various types of processing, including
proteolytic cleavage (snipping off of amino acids) and addition of chemical
modifications, such as phosphate groups.
All these steps (if applicable) need to be executed for a given gene for an active protein
to be present in the cell.
.
For instance, humans and chimpanzees have genomes that are about 98.8\% identical at
the DNA level.
Transcription factors are proteins that help turn specific genes "on" or "off" by binding
to nearby DNA.
 Transcription factors that are activators boost a gene's
transcription. Repressors decrease transcription.
 Groups of transcription factor binding sites called enhancers and silencers can turn a
gene on/off in specific parts of the body.
 Transcription factors allow cells to perform logic operations and combine different
sources of information to "decide" whether to express a gene.
Transcription factors are proteins that regulate the transcription of genes—that is, their
copying into RNA, on the way to making a protein.
RNA polymerase can attach to the promoter only with the help of proteins
called basal (general) transcription factors. They are part of the cell's core
transcription tool kit, needed for the transcription of any gene.

RNA polymerase binds to a promoter with help from a set of proteins called general
transcription factors.
However, many transcription factors (including some of the coolest ones!) are not the
general kind. Instead, there is a large class of transcription factors that control the
expression of specific, individual genes. For instance, a transcription factor might
activate only a set of genes needed in certain neurons.
How do transcription factors work?
A typical transcription factor binds to DNA at a certain target sequence. Once it's bound,
the transcription factor makes it either harder or easier for RNA polymerase to bind to
the promoter of the gene.

cis-acting elements
– Core Promoter – Basal level expression
• Binding site for TATA-binding protein and associated factors
– Promoter Proximal Elements - True level of expression
• Binding sites for transcription factors
Enhancers are another cis-acting element.
• They are required for maximal transcription of a
gene.
! Enhancers can be upstream or downstream of the transcription
initiation site
! They may modulate from a distance of thousands of base pairs away
from the initiation site.
! Enhancers contain short sequence elements, some similar to
promoter sequences.
! Activators bind these sequences and other protein complexes form,
postulated to bring the enhancer complex close to the promoter and
increasing transcription.

Regulatory elements that map near a

gene are cis-acting DNA sequences
 How do Enhancers work
if they are so far
away from the promoter?
 Possible looping of DNA
• Brings transcription factors together
 Transcriptional activators bind to specific promoters and enhancers at specific times to
Increase transcriptional levels

Activators
Some transcription factors activate transcription. For instance, they may help the
general transcription factors and/or RNA polymerase bind to the promoter, as shown in
the diagram below.

Diagram of an activator attached to a specific DNA sequence that is its binding site. The
other end of the transcriptional activator (the one not bound to the DNA) interacts with
general transcription factors, helping the general transcription factors and polymerase
assemble tat the nearby promoter.

Repressors
Other transcription factors repress transcription. This repression can work in a variety
of ways. As one example, a repressor may get in the way of the basal transcription
factors or RNA polymerase, making it so they can't bind to the promoter or begin
transcription.

When bound to this site, the repressor blocks formation of the transcription initiation
complex at the promoter of a nearby gene.

Binding sites
 The binding sites for transcription factors are often close to a gene's promoter. However,
they can also be found in other parts of the DNA, sometimes very far away from the
promoter, and still affect transcription of the gene.

The parts of an activator protein: the DNA binding domain (which attaches to the
recognition site in the DNA) and the activation domain, which is the "business end" of
the activator that actually promotes transcription, e.g., by facilitating formation of the
transcription initiation complex.

Turning genes on in specific body parts

Some genes need to be expressed in more than one body part or type of cell. For
instance, suppose a gene needed to be turned on in your spine, skull, and fingertips, but
not in the rest of your body. How can transcription factors make this pattern happen?
A gene with this type of pattern may have several enhancers (far-away clusters of
binding sites for activators) or silencers (the same thing, but for repressors). Each
enhancer or silencer may activate or repress the gene in a certain cell type or body part,
binding transcription factors that are made in that part of the body

Regulation after transcription

Alternative splicing, miRNAs and siRNAs, translation initiation factors, & protein
modifications.
Key points:
 RNA processing, such as splicing, capping, and poly-A tail addition
 Messenger RNA (mRNA) translation and lifetime in the cytosol
 Protein modifications, such as addition of chemical groups
In the sections below, we’ll discuss some common types of gene regulation that occur
after an RNA transcript has been made.

Regulation of RNA processing

When a eukaryotic gene is transcribed in the nucleus, the primary transcript (freshly
made RNA molecule) isn't yet considered a messenger RNA. Instead, it's an "immature"
molecule called a pre-mRNA.
The pre-mRNA has to go through some modifications to become a mature mRNA
molecule that can leave the nucleus and be translated. These include splicing, capping,
and addition of a poly-A tail, all of which can potentially be regulated – sped up, slowed
down, or altered to result in a different product.

Alternative splicing
Most pre-mRNA molecules have sections that are removed from the molecule,
called introns, and sections that are linked or together to make the final mRNA,
called exons. This process is called splicing. Post-transcriptional modification or Co-
transcriptional modification is the process in eukaryotic cells where primary
transcript RNA is converted into mature RNA. A notable example is the conversion
of precursor messenger RNA into maturemessenger RNA (mRNA) that occurs prior to
protein translation. The process includes three major steps: addition of a 5' cap, addition
of a 3' poly-adenylation tail, and splicing. This process is vital for the
correct translation of the genomes of eukaryotesbecause the initial precursor mRNA
produced during transcription contains both exons (coding or important sequences
involved in translation), and introns (non-coding sequences).[1]

• The modification is very much essential to eukaryotic systems.

Post transcriptional
•

modification includes
-> Capping at the 5'- end.
-> Tailing at the 3'- end.
-> mRNA splicing.

Capping at the 5'- End :

• 7- methyl guanosine is added at the 5’ end of almost all eukaryotic RNAs by a 5’-5’
triphosphate linkage.

• The capping process occurs in nuclei, prior to the splicing. m7GpppGp----

• The 5’cap protects the RNA from ribonucleases.

• The cap structure of mRNA will be recognized by the cap-binding protein required
for translation.

• The cap binds to cap binding complex of proteins which promotes binding of the
mRNA to the ribosome to initiate the process of translation.

• Generation of the 5’ cap occurs in a series of three to four steps shown below.
Poly-A Tail at 3' -END :

• Eukaryotic RNAs posses a string of 80 to 250 A residues at the 3’ end.

• This poly-A tail protects mRNA from enzymatic destruction, also acts as binding
site for many proteins.

• There is no poly-T sequence on the DNA template from which the RNA is
transcribed. The tailing process does not depend on the template.

• The tailing process occurs in the nuclei, prior to the splicing.

• The tailing process takes place in multiple steps with the help of the enzyme
polyadenylate polymerase.

• RNA + nATP -----> RNA-(AMP)n + nPPi (n = 80 - 250).

The processive polyadenylation complex in the nucleus of eukaryotes works on

products of RNA polymerase II, such as precursor mRNA. Here, a multi-protein
complex cleaves the 3'-most part of a newly produced RNA and polyadenylates the
end produced by this cleavage. The cleavage is catalysed by the enzyme CPSF and
occurs 10–30 nucleotides downstream of its binding site.[19] This site often has the
polyadenylation signal sequence AAUAAA on the RNA, but variants of it that bind
more weakly to CPSF exist.[18][20] Two other proteins add specificity to the binding to
an RNA: CstF and CFI. CstF binds to a GU-rich region further downstream of CPSF's
site.[21] CFI recognises a third site on the RNA (a set of UGUAA sequences in
mammals[22][23][24]) and can recruit CPSF even if the AAUAAA sequence is
missing.[25][26] The polyadenylation signal – the sequence motif recognised by the
RNA cleavage complex – varies between groups of eukaryotes. Most human
polyadenylation sites contain the AAUAAA sequence,[21] but this sequence is less
common in plants and fungi.[27]
The RNA is typically cleaved before transcription termination, as CstF also binds to
RNA polymerase II.[28] Through a poorly understood mechanism (as of 2002), it
signals for RNA polymerase II to slip off of the transcript.[29] Cleavage also involves
the protein CFII, though it is unknown how.[30] The cleavage site associated with a
polyadenylation signal can vary up to some 50 nucleotides.[31]
When the RNA is cleaved, polyadenylation starts, catalysed by polyadenylate
polymerase. Polyadenylate polymerase builds the poly(A) tail by adding adenosine
monophosphateunits from adenosine triphosphate to the RNA, cleaving
off pyrophosphate.[32] Another protein, PAB2, binds to the new, short poly(A) tail and
increases the affinity of polyadenylate polymerase for the RNA. When the poly(A)
tail is approximately 250 nucleotides long the enzyme can no longer bind to CPSF
and polyadenylation stops, thus determining the length of the poly(A) tail.[33][34] CPSF
is in contact with RNA polymerase II, allowing it to signal the polymerase to
terminate transcription.[35][36] When RNA polymerase II reaches a "termination
sequence" (TTATTT on the DNA template and AAUAAA on the primary transcript),
the end of transcription is signaled.[37] The polyadenylation machinery is also
physically linked to the spliceosome, a complex that removes introns from RNAs.[26]
Proteins involved:[12][18]
 CPSF: cleavage/polyadenylation specificity factor
CstF: cleavage stimulation factor
PAP: polyadenylate polymerase
PABII: polyadenylate binding protein 2
CFI: cleavage factor I
CFII: cleavage factor II

mRNA Splicing :
• The matured mRNAs are much shorter than the DNA templates.

• The mRNA which has been transcribed contains sequences encompassing one gene.
However, the structural genes are composed of coding and non-coding regions that
are alternatively separated. Hence, these are also termed as “split genes”.

• Exons are the coding sequences that appear on split genes and primary transcripts,
and will be expressed to matured mRNA.

• Introns are the non-coding sequences that are transcripted into primary mRNAs.

• During splicing, the introns are selectively removed from the primary transcript
while the exons are joined together to generate a continuous sequence that will code
entirely for a functional protein.

Spilicing is attained by different mechanisms :

Each spliceosome is composed of five small nuclear RNAs (snRNA) and a range of
associated protein factors. When these small RNAs are combined with the protein
factors, they make RNA-protein complexes
called snRNPs (small nuclear ribonucleo proteins, pronounced "snurps"). The
snRNAs that make up the major spliceosome are named U1, U2, U4, U5, and U6, so-
called because they are rich in uridine, and participate in several RNA-RNA and
RNA-protein interactions.

In the process of alternative splicing, different portions of an mRNA can be selected

for use as exons. This allows either of two (or more) mRNA molecules to be made from
one pre-mRNA.
Pre-mRNA Slicing Process
Splicing requires there to be three sequences in the introns. One end of the intron is the
5' splice site and the other end is the 3' splice site. At these sites are short consensus
sequences. The sequence that exons are ordered in the mRNA usually correlates with the
sequence in the corresponding DNA. The process is aided by spliceosomes, which are
small RNA molecules that recognize the beginning of introns (usually GU) and the end
(usually AG) and catalyze splicing at these sites. Changing a single nucleotide at these
sites may prevent splicing to occur. There are also self-splicing introns. The third
sequence important to splicing is located at the branch point. The branch point is where
an adenine nucleotide lies from 18 to 40 nucleotides before the 3' splice site. The
deletion or mutation of the adenine nucleotide at the branch point would prevent
splicing. Splicing occurs in large structures called spliceosomes.
Before splicing takes place, an intron between exon 1 and exon 2. Pre-mRNA splices in
two steps. In step one, the pre-mRNA is spliced at the 5' splice site, separating exon 1
from the intron. The 5' end of the intron then attaches to the branch point folding back
on itself and forming a structure called a lariat. The folding back occurs by the guanine
nucleotide in the 5' consensus sequence bonding with the adenine nucleotide at the
branch point through transesterification. In step two a splice is made at the 3' splice site
and the 3' end of exon 1 is attached to the 5' end of exon 2. The intron is separated as a
lariat and becomes linear when the bond breaks at the branch point and is then degraded
by nuclear enzymes. And finally, the mature mRNA consisting of only the exons spliced
together are moved to the cytoplasm and translated.
It is important to note that the 5' cap greatly affects pre-mRNA processing and mRNA
export and if it were ever to be removed, then it would be known as the first irreversible
step in mRNA decay which will affect the entire gene expression.
Before splicing takes place, an intron between exon 1 and exon 2. Pre-mRNA splices in
two steps. In step one, the pre-mRNA is spliced at the 5' splice site, separating exon 1
from the intron. The 5' end of the intron then attaches to the branch point folding back
on itself and forming a structure called a lariat. The folding back occurs by the guanine
nucleotide in the 5' consensus sequence bonding with the adenine nucleotide at the
branch point through transesterification. In step two a splice is made at the 3' splice site
and the 3' end of exon 1 is attached to the 5' end of exon 2. The intron is separated as a
lariat and becomes linear when the bond breaks at the branch point and is then degraded
by nuclear enzymes. And finally, the mature mRNA consisting of only the exons spliced
together are moved to the cytoplasm and translated.
It is important to note that the 5' cap greatly affects pre-mRNA processing and mRNA
export and if it were ever to be removed, then it would be known as the first irreversible
step in mRNA decay which will affect the entire gene expression.
CstF (CstF, cleavage stimulatory factor),is recruited by cleavage and polyadenylation
specificity factor (CPSF) and assembles into a protein complex on the 3' end to promote
the synthesis of a functional polyadenine tail, which results in a mature mRNA molecule
ready to be exported from the cell nucleus to the cytosol for translation.

RNA interference (RNAi) is a process in living cells. It

adjusts (moderates) the activity of their genes. RNAi molecules are a key to gene
regulation. In 2006, Andrew Fire and Craig Mello shared the Nobel Prize in Physiology
or Medicine for their work on RNA interference in the nematode worm Caenorhabditis
elegans, published in 1998.
Two types of small RNA molecules – microRNA (miRNA) and small interfering
RNA (siRNA) – do the work. These small RNAs bind to normal messenger
RNA (mRNA) molecules and increase or decrease their activity. They can prevent a
mRNA from producing a protein. RNA interference defends cells against foreign
nucleotide sequences – viruses and transposons. Also, they control development,
and gene expression in general.
The RNAi pathway is found in many eukaryotes including animals.[1] RNAi is a
valuable research tool, in cell culture and in living organisms. Synthetic dsRNA
introduced into cells can suppress specific genes of interest. RNAi may be used for
large-scale screens that shut down each gene to analyse cellular process or cell division.
The pathway is also used as a practical tool in biotechnology and medicine.

Natural functions
The natural functions of RNA interference are: [2][3]

1. Immunity against foreign virus (and other) RNAs

2. Upregulation of genes
3. Downregulation of genes

Small regulatory RNAs

Once an mRNA has left the nucleus, it may or may not be translated many times to
make proteins. Two key determinants of how much protein is made from an mRNA are
its "lifespan" (how long it floats around in the cytosol) and how readily the translation
machinery, such as the ribosome, can attach to it.
A recently discovered class of regulators, called small regulatory RNAs, can control
mRNA lifespan and translation.
 microRNAs
microRNAs (miRNAs) were among the first small regulatory RNAs to be discovered.
A miRNA is first transcribed as a long RNA molecule, which forms base pairs with
itself and folds over to make a hairpin.

First, a microRNA precursor is transcribed from a microRNA gene. The precursor folds
into a hairpin, which is then processed by enzymes so it is as short duplex (double-
stranded) RNA that's imperfectly complementary. One strand of this duplex is the
miRNA, which associates with a protein to form an miRNA-protein complex.
The miRNA directs the protein complex to mRNAs that are partially or fully
complementary to the miRNA. When the miRNA is perfectly complementary to the
mRNA, the mRNA is often cut in two by an enzyme in the protein complex. When the
miRNA is not perfectly complementary to the mRNA, the miRNA-protein complex may
remain bound to the mRNA and block translation.
The miRNA directs the protein complex to "matching" mRNA molecules (ones that
form base pairs with the miRNA). When the RNA-protein complex binds^22start
superscript, 2, end superscript:
 If the miRNA and its target match perfectly, an enzyme in the RNA-protein complex
will typically chop the mRNA in half, leading to its breakdown.
 If the miRNA and its target have some mismatches, the RNA-protein complex may
instead bind to the mRNA and keep it from being translated.
These are not the only ways that miRNAs inhibit expression of their targets, and
scientists are still investigating their many modes of action^33start superscript, 3, end
superscript.

Regulation of translation
We already saw how miRNAs can inhibit translation, but there are a number of other
ways that translation of an mRNA can also be regulated in a cell. One key step for
regulation is translation initiation.
In order for translation to begin, the ribosome, an RNA-and-protein complex that houses
translation, must assemble on the mRNA. This process involves many “helper” proteins,
which make sure the ribosome is correctly positioned. Translation can be regulated
globally (for every mRNA in the cell) through changes in the availability or activity of
the “helper” proteins.

For example, in order for translation to begin, a protein called eukaryotic initiation
factor-2 (eIF-2) must bind to a part of the ribosome called the small subunit. Binding of
eIF-2 is controlled by phosphorylation, or addition of a phosphate group to the protein.
When eIF-2 is phosphorylated, it's turned "off"—it undergoes a shape change and can no
longer play its role in initiation, so translation cannot begin. When eIF-2 is not
phosphorylated, in contrast, it's "on" and can carry out its role in initiation, allowing
translation to proceed.

In this way, phosphorylation of eIF-2 acts as a switch, turning translation on or off.

Inactivation of translation can be a good strategy in periods when the cell can't “afford”
to make new proteins (e.g., when the cell is starved for nutrients)^55start superscript, 5,
end superscript.

Proteins can be regulated after translation

There are also regulatory mechanisms that act on proteins that have already been made.
In these cases, an "edit" to the protein – such as removal of amino acids, or addition of a
chemical modification – can lead to a change in its activity or behavior. These
processing and modification steps can be targets for regulation.
For example, some proteins must be proteolytically cleaved (chopped up) in order to
become active. The insulin used by diabetics is one example. Other proteins may have
chemical groups added to them, including methyl, phosphate, acetyl, and ubiquitin
groups. Often, these groups can be added and removed dynamically to control activity.
Addition or removal of chemical groups may regulate protein activity or the length of
time a protein remains in the cell before it undergoes "recycling." Sometimes, chemical
modifications can also determine where a protein is found in the cell—for example, in
the nucleus or cytoplasm, or attached to the plasma membrane.

Phosphorylation
One of the most common post-translational modifications is phosphorylation, in which
a phosphate group is attached to a protein. The effect of phosphorylation varies from
protein to protein: some are activated by phosphorylation, while others are deactivated,
and others yet simply change their behavior (interacting with a different partner, or
going to a different part of the cell).

Image of a protein with a phosphate group attached, showing the chemical structure of
the phosphate group, which bears a negative charge.
We saw one example of this above, when we examined how eIF-2 is inactivated by
addition of a phosphate group (blocking translation). However, many different proteins
can be selectively phosphorylated, producing various effects depending on the protein's
role in the cell.

Ubiquitination
Proteins can be tagged for degradation by the addition of a chemical marker
called ubiquitin. Ubiquitin-tagged proteins are taken to the proteasome, or “recycling
center” of the cell, and broken down into their component parts. Ubiquitination is an
important way of controlling the persistence of a protein in the cell.

How a protein is tagged with ubiquitin and degraded. First, ubiquitin is attached to the
protein. Then, the protein is taken to the proteasome, where it is broken down and
"recycled" for parts.