Eukaryotic genes

• Eukaryotic genes also are regulated by protein-coding

sequences and control elements, but no operons

• Eukaryotic gene regulation is more complex because

eukaryotes possess a nucleus.

• (transcription and translation are not coupled).

 Eukaryotic Chromosome
• Chromosomes – tightly coiled DNA
around proteins during cell division
• Chromatin – loosely packed DNA
around proteins
• Histones – protein which the DNA
wraps around
• Nucleosomes – grouped histones
together
o Heterochromatin – tighter packed
chromatin
• Not transcribing
o Euchromatin – looser packed
chromatin
• Transcription occurring
 Nucleosome Structure

Nucleosomes contain 2 copies

of H2A, H2B, H3 and H4

147 bp of DNA is wrapped

around nucleosome

H1 is a linker histone

from Jiang and Pugh, Nature Rev.Genet. 10, 161 (2009)

Two “categories” of eukaryotic gene regulation
exist:

o Short-term - genes are quickly turned on or off in

response to the environment and demands of the cell.

o Long-term - genes for development and differentiation.

Control of Gene Expression

39
Eukaryote gene expression is regulated at seven levels:

1. Gene Regulation at DNA Level

2. Transcription

3. RNA processing

4. mRNA transport

5. mRNA translation

6. mRNA degradation

7. Protein degradation
1. Gene Regulation at DNA Level
 Chromatin Remodeling

1. Changes of DNA Topo structure. Formation of ssDNA

 What does “Epigenetics” mean?

• Gene expression changes not caused by changes in DNA sequence

• Genes are “turned on” or “turned off” by chemical alterations to:

- DNA (Methylation)
- Histone modification (methylation, acetylation, etc.)

• Epigenetic changes can be heritable

• If DNA (genome) is the hardware of a computer, epigenetics

(epigenome) is like the “software”.

• Provides an additional layer of information on the genetic info.

2. DNA Methylation

DNA Methylation
 At CpG islands
----- are genomic regions that contain a high
frequency of CG dinucleotides.

----- CpG islands particularly occur at or near

the transcription start site (+1) of housekeeping
genes.
 TF RNA pol

Active
transcription
Unmethylated CpG island

TF RNA pol

CH3 CH3 CH3 Repressed

transcription

Methylated CpG island

3. Histone modification
 methylation
 acetylation

TF
Chromatin Regulation
• Histone acetylation – allows
transcription factors to bind to DNA
allowing transcription to occur
o Creates loosely packed DNA –
euchromatin

• DNA Methylation – occurs after

DNA synthesis has occurred
o Lower transcription rates
o One X in females is highly
methylated
Properties of Acetylated Histones

 Less positively charged

 Chromatin is less condensed

DNA Methylation & Histone Modifications
Form the Epigenetic Code
 Histone Modifications Affect Chromatin Structure

H3K4 methylation and H3K9 acetylation

are hallmarks of active chromatin

H3K27 methylation and H3K9 methylation

are hallmarks of silent chromatin

from Johnstone and Baylin, Nature Rev.Genet. 11, 806 (2010)

H: Histidine, K: Lysine
 Epigenetic inheritance
• Not controlled by base sequences.

• DNA methylation (deactivates one

homologous chromosome) may explain
abnormal or unexpected DNA expression
as is often seen in identical twins.
4. Other gene regulation at DNA Level
(1) Gene Deletion
(2) Gene Duplication
(3) DNA Rearrangement
(4) Gene Amplification
(5) Chemical Activator
(6) Environmental Activator
2. Transcription
Promoter sequence
Promoters
• Occur upstream of the transcription start site (+1)
 Core promoter
 in eukaryote: TATA-box: determine where
transcription begins
 in prokaryote: -10 region

 Promoters are activated by specialized transcription

factor (TF) proteins
 Promoters may be positively or negatively regulated.
Enhancers

 Occur upstream or downstream of the transcription start site.

 A regulatory DNA sequence that greatly enhances the transcription

of a gene.

In eukaryotes, enhancers may be very distant from the promoter.

 Gene expression

 Constitutive gene expression in all cells

(housekeeping genes)

 Induced genes whose expression is regulated by

environmental, development or metabolic
signals
 Regulation of Transcription

• Enhancer – control element far from a gene or intron

• Activator – bind to enhancers to turn on transcription of a
gene
• Transcription factors + enhancer + activator + RNA
Polymerase II = transcription initiation complex
o Needed for transcription to begin

• Repressors – inhibit gene expression

o Turn off transcription
o Block activators from binding to enhancers
 Distal control
element
Promoter
Activators
Gene

Enhancer TATA
box
1 General
Activator proteins bind transcription
to distal control elements factors
grouped as an enhancer in
the DNA. This enhancer has
three binding sites. DNA-bending
protein

2 Group of
A DNA-bending protein Mediator proteins
brings the bound activators
closer to the promoter.
Other transcription factors,
mediator proteins, and RNA RNA
polymerase are nearby. Polymerase II

Chromatin changes

3
The activators bind to Transcription

certain general transcription RNA processing

factors and mediator RNA

proteins, helping them form Polymerase II
an active transcription mRNA
degradation
Translation

initiation complex on the promoter.

Protein processing
and degradation

Transcription RNA synthesis

Initiation complex
 RNA Polymerase Binding to Promoters Is a

major Target of Regulation

• RNA polymerases bind to promoter sequences near

the starting point of transcription initiation.

• The RNA pol-promoter interaction greatly

influences the rate of transcription initiation.
 TF RNA pol

Active
transcription
Unmethylated CpG island

Regulatory proteins (transcription factors) work to enhance or

inhibit this interaction between RNA pol and the promoter DNA.
 Small-Molecule Effectors Can Regulate
Activators and Repressors
• Repressors reduce RNA Pol-promoter interactions or
block the polymerase.
o bind to operator sequences on DNA
• usually near a promoter in bacteria but further away in many eukaryotes
 Negative Regulation
• Negative regulation involves repressors.
o Example: Repressor binds to DNA and shuts down
transcription
o Alternative: Signal causes repressor to dissociate from
DNA; transcription induced

Despite opposite effects on

transcription, both are negative
regulation
 Positive Regulation
• Positive regulation involves activators.
• Enhance activity of RNA polymerase

• Activator-binding sites are near

promoters that weakly bind
RNA Pol or do not bind at all.
DNA looping allows eukaryotic enhancers to
be far from promoters
• Activators can influence transcription
at promoters thousands of bp away.

• How? Via formation of DNA loops

• Looping can be facilitated by

architectural regulator proteins.

• Co-activators may mediate binding

by binding to both activator and RNA
polymerase.
More about promoters and enhancers:
• Some regulatory proteins (TF) 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.
4. Terminator
A DNA sequence just downstream of the coding
segment of a gene, which is recognized by RNA
polymerase as a signal to stop transcription.

5.Silencer
A DNA sequence that helps to reduce or shut off the
expression of a nearby gene.
Chromatin remodeling
Acetylation of histones
enhances access to
promoter region and
facilitates transcription.
Short-term - transcriptional control of
galactose-utilizing genes in yeast
Galactose metabolizing pathway of yeast.

GAL1
galactokinase

GAL7
galactose transferase • In absence of
GAL10
galactose, GAL
galactose epimerase genes are not
transcribed.

• GAL genes rapidly

induced by
galactose and
absence of glucose.

• Analagous to E. coli
lac operon
repression by
glucose.
Short-term - transcriptional control of
galactose-utilizing genes in yeast

• A repressor protein that binds a promoter element

called an upstream activator sequence (UASG).

• Transcription occurs in both directions from UASG.

Activation model of GAL genes in yeast.
 Gene  Protein Control
• Feedback inhibition – enough
product is made the system shuts
down
o More product is made when
needed
o The product shuts down the
process

• Gene Expression – genes are

only expressed when needed.
Often regulated at transcription.
Trp operon: repressible, always
making tryptophan, repressed if
tryptophan is “eaten”
tryptophan is necessary for the
cell to function
Micro-RNAs prevent translation of mRNA

• Micro-RNAs (miRNAs) silence genes by binding to mRNAs

o can prevent transcription of the mRNA by cleaving it (via
endonucleases Drosha or Dicer) or by blocking it

• Some miRNAs are made briefly during development; they are

called small temporal RNAs (stRNAs).
MiRNA
• miRNA’s - micro RNA can
degrade mRNA or block
translation
• Causes mRNA to fold on
itself and base pair to create
dsRNA which is then
digested with an enzyme
• Short interferring RNA
(siRNA) – also degrade
mRNA or block translation
(blocking by siRNA is called
RNAi, or RNA interferance)
 Researchers can shut down genes
artificially via RNA interference

• Any dsRNA that corresponds to an mRNA and is introduced

into a cell will be cleaved by Dicer into short segments called
small interfering RNAs (siRNAs).

• These will bind to the mRNA to silence its translation.

• The process is called RNA interference.

Gene Silencing by RNA Interference
3. RNA processing control
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 spliced

• Alternative polyadenylation and splicing can occur together.

 Expression of a Typical Eukaryotic Gene
Protein Coding Gene

…
DNA
Transcription Polyadenylation
exon intron

primary transcript / pre-mRNA

Splicing

AAAAAAAAA
mRNA Export
Translation Degradation

Protein
Folding, Modification,
Transport, Complex
Assembly
Protein Complex
Degradation
93
Introns are removed by a process called splicing
Alternative polyadenylation and splicing of the human
CACL gene in thyroid and neuronal cells.

Calcitonin gene-
related peptide
Alternative splicing in sex
determination of Drosophila

•Sex is determined by X:A

ratio.

•Sxl (sex lethal) gene

determines the pathways for
males and females.

•If X:A = 1, all introns and

exon 3 (which contains the
stop codon) are removed.

•If X:A = 0.5, no functional

protein is produced.
4. mRNA transport control
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.

5. mRNA translation control
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.
cytoplasmic polyadenylate-binding protein (PABPC)
 Eukaryotic mRNA deadenylation and decay. Deadenylation by exonucleases PAN2–PAN3
and CCR4–NOT
Lori A. Passmore et al., 2022
6. mRNA degradation control
6. 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

7. Post-translational control - protein degradation
7. 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
 Protein processing
- Inactive form cut to become active (Pro-insulin)
Summary and contrasts:

Prokaryotes control expression by:

Transcription

Eukaryotes control expression by:

Transcription

RNA processing

mRNA transport

mRNA translation

mRNA degradation

Protein degradation
Targets of Regulation in Eukaryotes
In order for a specific gene to be transcribed, its regulatory sequences must be exposed by
chromatin remodeling factors that move regulatory sequences out of nucleosomes, so that they
can be recognized by the protein complex that initiates transcription.
Once regulatory sequences have been exposed, the basal transcription
complex assembles over the promoter, while regulatory transcription
factors bind enhancers
RNA polymerase is then free to join the transcription complex and initiate transcription
Gene Regulation & Cancer
1. Features of Cancer Cells
2. Causes of Cancer:
 Viruses
 Tobacco smoke
 Food
 Radiation
 Chemicals
 Pollution
3. Proto-oncogene & Oncogene

 Proto-oncogene - is a normal gene that can

become an oncogene due to mutations or
increased expression.
 Oncogene - is a protein encoding gene, which -
when deregulated - participates in the onset and
development of cancer.
 Tumour suppressor gene - or antioncogene is a
gene that protects a cell from being cancer.
4. Proto-oncogene activation
 5. p53 and cancer

 It can activate DNA repair proteins when DNA has

sustained damage.

 It can also hold the cell cycle at the G1/S regulation point
on DNA damage recognition

 It can initiate apoptosis, the programmed cell death, if

the DNA damage proves to be irreparable.