Transcript Eukaryotic Gene Regulation

Eukaryotic Gene Regulation
Introduction
• Difference between eukaryotic and
prokaryotic DNA
• Regulation at chromosome level
• Regulation at the transcription level
• Post transcriptional regulation
– Alternative Splicing
– microRNA…
• Post-translation modification
Prokaryotic DNA V Eukaryotic DNA
• Prokarytoic is circular, eukaryotic is straight.
• Eukarytoic DNA is in the form of chromatin [prokaryotic is
not]; essentially Eukaryotic is surrounded by a histone
envelope.
• The promoter region of a prokaryotic gene [coding region]
is immediately “upstream” of the gene. In eukaryotic DNA
there are more that one promoter region which can be a
large distance from the gene
• Prokaryotic genes are in the form of operons[polycistronic];
e.g. lac operon, while eukaryotic normally are associated
with one gene and are regulated by silencers and
enhancers. These enhancers can be controlled by more
that one regulatory element [trans]
• The coding region of Eukaryotic DNA consists of exons [
regions that are translated] interjected with introns
[regions that are not translated] . Prokaryotic coding
regions do not have introns;
Overview of Eukaryotic gene regulation
level 1: Regulation at the chromatin level
• Histones are proteins that surround and “protect” DNA
and form chromatin
• While the histones conceal the DsDNA so no RNA/DNA
polymerase can bind to it.
• Chromatin modification can be considered to be the
first step of gene regulation:
– Prerequisite for some gene(s) transcription
– Simultaneous with others [dna exposed and then
transcribed]
• Forms the basis of the field of epi-genetics:
modification of the phenotype with any change to the
genotype or DNA sequences.
level 2: Expression Ctrl at the transcription level
• Much 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
level 2: Expression Ctrl at the transcription level
Ref [1] p 321
The Core promoters regions :
Just upstream of where RNA polymerase binds and
transcription starts [transcription start site]
initiating low level transcription;
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 at some distance
on either side of the gene or within it
– Required to achieve maximum level of expression
– There position is not fixed and 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)
– Time and tissue specific (play a part in organism
development.
level 2: Expression Ctrl at the transcription level
• Silencers :
– Cis-acting transcription regulatory element
– Acts upon the gene to repress the level of transcription
that was initiated by the corresponding promoter.
– Are tissue specific and temporoal-specific
– 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: Post transcription regulation
• Alternative splicing:
• 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
• However, for many genes the introns can be spliced in more
a number of ways or 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].
• The are a number of splicing process and it is regulated like
DNA transcription
Illustration of Alternative splicing
Adapted from [3]
•
•
40-60% of genes have alternative splicing forms.
Frequencies of splices can vary 1 - thousands.
– Encoding proteins at nodes highly connected
interaction networks e.g. neural tissue
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.
2. a different translation termination site by the addition/removal of a stop
codon in the coding sequence (alternative termination).
Note a poly A tail 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 to the
ribosomes. .
3.
Alternative splicing can also change the internal region because of an inframe insertion or deletion.
AS regulation[2]
Normal
regulation
Cis-acting
splicing
disorder
Indirect
Trans-acting
splicing
disorder
Examples of abnormal A.S. regulation [2]
• Cis-acting disorder:
– Found in neurological disease such as spinal
muscular atrophy
• Indirect transacting-acting disorder:
– Found in Prader Willi syndrome; ocd, autism
• Direct trans-acting disorder.
– Cases of epilepsy and mental retardation.
Level 5: Regulation via RNA degradation
• Small fragments of RNA strands called Micro RNA
(miRNA) (22 nucleotides in length). Can regulated
gene expression in a number of ways:
– Degrade the target (mature) mRNA
– Prevent the early stages on translation by ribosome
“drop off”
– Affect chromatin Remodelling by causing histones to
bind more tightly to the DNA and so prevent pretranslation expression. This process can hace a
significant affect as it can “knock out” large segments
of the DNA (100 to 1000s of genes
• An aside: It seems that the origins of the process
was protecting cells from viral infection.
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
Exam question
• A bacterial genome is different from a animal/plant
genome discuss how the effect of such differences on
the the regulation of gene expression in Eukaryotic
cells animal cells to be more complex.
• “Alternative splicing is a critical reason as to why the
genome of humans is much smaller than would be
expected”. Discuss how the alternative splicing causes
more proteins are produced than the number of genes
present in the genome of Eukaryotic cells.
•
References
• 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