Regulation of eukaryotic gene
sequence expression
Lecture 6
Introduction
• Difference between eukaryotic and
prokaryotic DNA
• Brief Overview of Eukaryotic “gene”
expression regulation.
• Regulation at the transcription level
• Post transcriptional regulation
– Alternative Splicing
• Pre/ Post-translation modification
Prokaryotic DNA & Eukaryotic DNA
• The following gives the broad differences
between the different cell classes:
1. Prokarytoic is circular, eukaryotic is straight.
2. Eukarytoic DNA is in the form of chromatin
[prokaryotic ]; essentially Eukaryotic is
surrounded by a histone envelope.
3. Prokaryotic genes are in the form of operons[
with polycistronic, multiple genes, mRNA]; e.g.
lac operon, while eukaryotic DNA is
monocistronic, one gene.
Prokaryotic DNA & Eukaryotic DNA
4. The promoter regulatory systems in
prokaryotic has a repressor gene sequence;
while in eukaryotic DNA there are enhancers
and silencers sequences.
5. The protein coding region of Eukaryotic DNA
consists of exons [ regions that are
translated] interjected with introns [regions
that are not translated]. In prokaryotic DNA
the sequences is continuous [ no introns]
Overview of Eukaryotic gene regulation
• Eukaryotic cells have more
than one level of
“transcription” control.
• The students can refer to
the supplementary lecture
for details on:
– Chromatin remodelling
(level 1)
– miRNA (level 5)
level 2: Expression Ctrl at the transcription level
• More complex than prokaryotic consists of:
• Promoter: like prokaryotics is the region where RNA
polymerase binds. [ refer to p region in the lac operon]
– There different promoter “regulatory” sites : e.g. core (basal
promoter), distal (upstream )promoter.
• Enhancers: regions that increase transcription levels
• Silencers: regions that decrease the level of transcription
• Both enhancers and silencers can be thousands of bp away
form the transcription site
• Similar to prokaryotic cells promoters have specific sequences
[refer to supplementary notes]
level 2: An eukaryotic promoter sequence
The Core promoters regions :
Just upstream of where RNA polymerase binds and
transcription starts [transcription start site]
Contains TATA and/or CAAT boxes and/or CG rich
level 2: Expression Ctrl at the transcription level
• Enhances:
– DNA sequences that can be located: upstream or
downstream of the “gene”
– Required to achieve maximum level of expression
– They seem to be generic to an extent (an enhancer
need not be gene specific ([1] p 322)
– They can also be inside the gene they regulate; Ig
heavy chain enhancer.
– Can enhance more than one gene; e.g. β and ε globins
in chickens (ref [1] p. 322)
– There activation is time and tissue specific (play a
part in organism development.)
level 2: Expression Ctrl at the transcription level
• Silencers :
– Repress the level of transcription that was initiated by the
corresponding promoter.
– Their location like enhancers can be in different locations.
– Are tissue specific and time-specific
– The following example illustrates how silencers and
enhancers work in tandem to control gene expression:
• E.g. found in gene that produces a hormone involved in thyroid
production/stimulation . This hormone is only produced in pituitary
cells. Expression only occurs in these cells because of a silencer that
binds a cellular factor which repress transcription. However, in cells
that are required to produce the hormone the effect of the silencer
is itself neutralised by an enhancer located 1.2 kb upstream of the
promoter of the gene and is only “activated” in the cells
[thyrotrophs] that must produce this hormone
Level 3: Exon and intron sequences
• The coding region [gene] of Eukaryotic DNA consists
of regions called exons interjected with introns.
• Prior to translation these introns must be “cut out”
spliced from the pre mRNA [ mRNA] to produce
mature mRNA
• The following figure shows the DNA sequence of a
eukaryotic gene “system” (in will be discussed in
more detail in the lecture: “Finding genes”
Level 3: Alternative splicing
• In 40% to 60% of genes the introns can be spliced in a number of
ways to produces alternative spliced mature mRNA strands. Only
mature mRNA strands are translated into amino acid strands.
• The consequence of this process [Alternative Splicing] is that one
DNA coding region can produce many mature mRNA strands and so
many proteins. With some genes being able to produce ~38,000
different mature mRNA strands [splices]. Mostly associated with
immunological proteins.
• Like DNA transcription RNA splicing is also regulated (refer to
supplementary notes.)
• Methods and computational techniques used to detect splices can
be found in Alternative splicing: global insights [not required
reading! But may prove interesting]
Level 3: Basic Alternative splicing
Adapted from [3]
• The above illustrate what is referred to as (mutually exclusive)
splicing.
• There are four possible types of splicing.
Types of Alternative splicing
A more comprehensive description A.S. can be found at ref [5 and 6]
Alternative splicing: the effects
• Alternative splicing can lead to:
1. use of a different site for translation initiation (alternative initiation);
alternative promoter/exon.
What might this mean in relation to the DNA sequence?
What does it suggest about the possible location of promoters?
1. a different translation termination site by the addition/removal of a
stop codon in the coding sequence (alternative termination).
3.
Alternative splicing can also change the internal region because of an
in-frame insertion or deletion.
Level 4: The CAP and Poly A tail
• After splicing the mRNA undergoes two other changes. These can be used
to distinguish pre-mRNA form mature mRNA
•
•
The poly A tail, polyadenylation, is a sequence of adenine (A) RNA molecules added to
the end (3’ end) of the mature mRNA.
In addition a “CAP” complex [modified G RNA molecule] is added to the 5’ start of the
mature mRNA;
• both play a part in protecting the mRNA from degradation while it is being
transported into the cytoplasm and to the ribosomes.
• Level 5 (Degradation of mRNA by microRNA strands) and level 6 and 7
(translational and post translational) can be looked at in supplementary
material
Level 6: Translation/post translational Modification
• The protein levels and activities can also be controlled via:
– Regulation of the protein stability and modification
– An example of such a protein is p53 a transcription factor for a
number of important cell cycle genes.
– Normally its low but in a damaged cell levels increase and helps
express these important genes
– When cells are damaged P53 Protein modification results in its
activation and furthermore the modification decrease its rate of
degradition.
– However P53 is controlled by a negative feedback loop so
eventually the levels go back to their original low levels ([1] p
327)
• Insulin is an example of a molecule that undergoes posttranslational modification. When it is translated it is in an
inactive form and modification is required to active it.
Exam question
a)
b)
c)
Prokaryotic DNA has a number of differences from Eukaryotic DNA
Describe three differences between each type of DNA (9 marks)
Explain how two of these differences result in extra steps in the
Eukaryotic gene regulation process (12 marks)
Explain, using a suitable example, how one of the extra regulatory
steps will affect Eukaryotic DNA sequence analysis. (9 marks)
• “Alternative splicing is a critical reason as to why the genome of
humans is much smaller than would be expected”.
1.
2.
3.
Explain how Alternative splicing ensures that the human geneome
does not have to be as large as might be expected. (5 marks)
Describe, using examples, 3 types of alternative splicing (15 marks)
Discuss the potential impact alternative splicing may have on the
analysis of Eukaryotic DNA coding sequences (10 marks).
References
• [1] Klug Essentials of Genetics: chapter 15 7th
Edition
• [2] Licatalosi, D.D. and Darnell, R.B. 2006.
Splicing Regulation in Neurologic Disease.
Neuron 52, 93-101
References
• [3]
http://www.ncbi.nlm.nih.gov/Class/MLACourse/
Modules/MolBioReview/alternative_splicing.html
• [4] Modrek, B. and Lee, C. 2002. A genomic view
of alternative splicing. Nature genetics 30, 13-19.
• [5]
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1
370565/
• [6] IntronsIntron Retention Retention