Transcript Regulation of Gene Expression

Molecular Biology
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Largely Concerned with Gene Expression
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What Turns it On/Off?
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How that is Achieved?
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How Much?
Regulation of Gene Expression - 2 lectures
In Eukaryotes Regulation of Gene Expression
is Complex - Not just on and off  Vary magnitude
Regulation of Gene Expression
1 - Revision of Eukaryotic Gene Structure
2- Need for Global regulation of genes
Molecular cell Biology 5th Edition
Mitochondria structure, aerobic and anaerobic metabolism
pp 171-172, 304-312
Gene structure
pp 405 -408
Molecular Genetic Techniques and Cloning
Chapter 9
Journal of Experimental Biology
Volume 201, pp 1177-1195 (1998)
Kwast et al.
Regulation of Gene
expression in Eukaryotes
Gene regulation in prokaryotes and/or
single cellular organisms is different in
that gene regulation is primarily
concerned with responding to external
stimuli. These include nutrients,
temperature.
Although multicellular organsims also
respond to such stimuli, eg plants,
another level of gene regulation is
involved.
For multicellular organisms the right
gene must be activated at the right time in
the right cell.
Examples of gene regulation at all levels
have been documented, transcriptional
Eukaryotic messages have 3’ untranslated
region
can also vary in size from tens to
hundreds of bases
Also have a poly A tail
A series of A residues that are added in a non
template dependant manner after
transcription
The amount of information in Eukaryotic
DNA is 1 X 109 bp
Estimated to be 35, 000 genes
The function of non-coding DNA is not
known, obviously some of the non-coding
DNA acts as “promoter” regions etc.
However still lots of DNA that appears to
have no apparent function.
Gene
- Is a piece of DNA that encodes a
functional unit
- remember have ribosomal and
tRNA genes so genes not restricted to
encoding proteins
Central Dogma of molecular biology is that
DNA is the genetic material that is replicated
and passed onto succeeding generations. It is
transcribed into RNA, which is subsequently
translated into protein.
RNA - Three types
Ribosomal RNA (rRNA) - as name
suggests found in ribosomes which
function to synthesise proteins
Messenger RNA (mRNA) - This type of
RNA specifies the sequence of amino acids
in a protein by triplet codon bases. The
mRNA sequence is translated into a
protein sequence.
Transfer RNA (tRNA) - This RNA acts as
an intermediate between the mRNA and
protein. Through complementary base
pairing to the mRNA it delivers the amino
acid coded in the mRNA to the ribosome.
Proteins - all proteins are encoded for by
mRNA and synthesised on ribosomes. These
have various functions including structural
(collagen, cell membrane proteins), enzymatic
(digestive, intracellular metabolism), signals
(insulin) and defence (antibodies).
Obviously get exception to this flow of
information
1) RNA viruses
2) RNA editing
3) Ribozymes
4) Prions
If RNA is reversed transcribed to produce
DNA, the DNA is called cDNA, as it is
complementary to the RNA copy.
No process of reverse translation known to
date
Relationship between the structure of protein,
mRNA and Genes
This is not the structural relationship
between Protein, mRNA and genomic DNA in
Eukaryotic cells
The relationship is far more complicated and
it is important to get the terminology correct
as you will come across it in texts etc.
Eukaryotic messages have a 5’ Cap
Also have a 5 ‘ untranslated region
can Vary from tens to hundreds of bases
Again this is not the relationship between
mRNA and genomic DNA
Genomic genes in Eukaryotes contain
intervening sequences known as Introns.
Therefore the initial RNA transcript contains
both exons and introns, the introns are
removed from the primary RNA transcript in
a process call splicing
How do we know where translation starts in
the mRNA
First in frame AUG
Not necessarily the first AUG but the first in
frame AUG
Find this from:
I) Protein sequence - care as lots of
eukaryotic proteins are processed
ii) Sequence analysis on a computer
How do we know where transcriptional
begins Transcriptional start site
Need to know before we can start about
promoter elements etc
Two methods
1) S1 nuclease mapping
2) Primer extension
Will use control of gene expression
by oxygen as an example
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Take place in all organisms
- fungi, plants and animals
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Critical for survival
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Evolved early so can use
comparative approaches
to Understand
Oxygen is Toxic
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Will Not deal with Reactive Oxygen Species - Rather will deal with
Role of oxygen in respiration - Oxidative Phosphorylation
Aerobic respiration essential for survival of multi-cellular
Organisms
Some micro-organisms can survive anaerobically
Anaerobic - yeast - Industrial applications
Plants - Loss of Yield - results in large loss of life
and/or commercial income
Animals - Medical conditions - hear disease, cancer etc
Genome Information
S. cerevisiae C. elegans
# Cells
Size
Chromosomes
Predicted ORFs
%Coding
A. thaliana
H. sapiens
~1000
>1x106
>1x106
12Mbp
97Mbp
125Mbp
3.2 Gbp
16
6
5
23
~6,000
~19,000
~28,000
~35,000
72%
27%
50%
1.5%
1
yeast carbon metabolism
Aerobic metabolism:
(oxidative metabolism)
Glycolysis TCA cycle oxidative phosphorylation
Anaerobic metabolism:
(fermentation)
Glucose  Acetyl CoA  Ethanol
Diauxic shift:
Metabolic change as fermentable carbon source is
used up from…
Glucose Fermentative
(Glycolysis  Ethanol)
to…
Oxidative Metabolism
(Ethanol  TCA cycle)
Mitochondrial structure
• Two membranes
• Inner membrane invaginated
• Numbers of mitochondria per cell
vary but usually 100s/cell
Matrix contains the TCA
cycle (and other) soluble
enzymes
Inner membrane
contains metabolite
transporters and the
electron transport chain
Overview of
aerobic
respiration
Four large, multi-subunit
protein complexes
- complex I is a NADHubiquinone reductase
- complex II is succinate
dehydrogenase (part of the
TCA cycle)
- complex III is the
ubiquinone -cytochrome c
reductase
- complex IV is cytochrome
oxidase
The respiratory electron transport chain
Outline of Tricarboxylic Acid Cycle
3-Carbons
One pyruvate molecule is
completely oxidised to CO2
CO2
4-Carbons
6-Carbons
NADH
NADH + CO2
FADH
NADH
+ CO2
The NADH and FADH
produced are oxidised
by the respiratory
electron transport chain
Mitochondria have their own DNA and Ribosomes
Mitochondria have some of their own DNA, ribosomes, and can make many
of their own proteins. The DNA is circular and lies in the matrix in structures
called "nucleoids". Each nucleoid may contain 4-5 copies of the
mitochondrial DNA (mtDNA).
mitochondrial
DNA
Organisation of the
mitochondrial chromosome
Human mtDNA
• small, double stranded
circular chromosome
• 16,569 bp in total
• no non-coding DNA
• no introns
• polycistronic replication
which is initiated from
the D (displacement)- loop
region
• followed by splicing of
transcript to form
messages.
Yeast
mitochondrial
chromosome
yeast mtDNA
human mtDNA
maize mitochondrial
genome
Synthesis of mitochondrial proteins
In all organisms, only a few of the proteins of the
mitochondrion are encoded by mtDNA, but the precise
number varies between organisms
• Subunits 1, 2, and 3 of cytochrome oxidase
• Subunits 6, 8, 9 of the Fo ATPase
• Apocytochrome b subunit of complexIII
• Seven NADH-CoQ reductase subunits (except in yeast)
The nucleus encodes the remaining proteins which are
made in the cytosol and imported into the mitochondrion.
Most of the lipid is imported.
Mitochondria are largely maternally inherited in higher
animals and plants
In mammals, most of the mitochondrial DNA (mtDNA) is
inherited from the mother. This is because the sperm
carries most of its mitochondria its tail and has only about
100 mitochondria compared to 100,000 in the oocyte.
Although sperm mitochondria penetrate the egg,
most are degraded after a few hours. As the cells
develop, more and more of the mtDNA from males
is diluted out. Hence less than one part in 104 or
0.01% of the mtDNA is paternal.
Mitochondria are largely maternally inherited in
higher animals and plants
This means that mutations of mtDNA are passed
from mother to child. It also has implications for the
cloning of mammals with the use of somatic
cells. The nuclear DNA would be from the donor cell,
but the mtDNA would be from the host cell. This is
how Dolly the sheep was cloned.
In plants, the cytoplasm, including the
mitochondria and the plastids, are contributed
only by the female gamete and not by the pollen again, mutations in organelle DNA are inherited
maternally.
Human Evolution and mtDNA
• Mitochondria divide by fission and are not made de novo
• they are inherited mainly from the mother:
>99% of our mitochondria are derived from those
(1000 or so) present in our mother’s ovum
Human Evolution and mtDNA
D-loop:
origin of mtDNA
replication
Human evolution can
be traced by analysis
of the base sequence
in a small part of the
mitochondrial genome
which does not encode
a gene and which is
quite variable.
- the so-called D-loop.
Human Evolution and mtDNA
The D-Loop of the mtDNA is the start of
replication/transcription site and contains 400-800
bp
Unlike the rest of mtDNA in humans, which is
highly conserved, this region is very variable
between people
It also has a very high frequency of change during
evolution (about 2% per million years)
Human Evolution and mtDNA
By comparing different groups, we can get a
glimpse of human evolutionary lines.
Eg, African individuals have more variability
between each other than do Asians, indicating
that the former have had more time to
accumulate changes - ie, the Africans are a
more ancient group.
Human Evolution and mtDNA
This makes the D-loop a very powerful tool for
the study of evolutionary relationships between
organisms and for DNA typing of individuals.
In addition, because of the large number of
mitos in a cell, extracting mtDNA is easier from
small amounts of tissue - and it can be readily
separated form other DNA by centrifugation on
CsCl gradients.
Extrapolating this in evolutionary terms, this
means that all mitochondria came from a “single”
ancestral female
- the so-called “Mitochondrial Eve”.
References:
Proceedings of National Academy Sci (USA) 91:8739 (1994)
Science 279: 28 (1998)
However, this is based on the assumption that mitochondrial
inheritance is strictly clonal. Recent evidence shows that mitos
from sperm do enter the egg and last for several hours. If
recombination occurs between mitos, then the Eve hypothesis
may be incorrect - or at least the timing would be incorrect.
Proc. R. Soc. Lond. B (1999) 266, 477-483
Human Evolution and mtDNA
But we have to be careful: the rate of change in
mtDNA may not be constant and heteroplasmy
(due to recombination of mtDNA) may cause
complications. Also, mtDNA represents a single
lineage and other genetic changes need to be
traced also.
However, when this was done with
polymorphisms in the Y chromosome, ‘Adam’
was also traced back to Africa, at about the
same period.
Human Evolution and mtDNA
Assuming that the rate of change in the D-loop is
constant and due only to mutation, the number of
difference s between Africans can be use to
calculate when their common ancestor lived. This
works out to be about 200,000 years ago.
This suggests that modern Homo sapiens came out
of Africa at about that time and migrated through
Europe and Asia, replacing other early humans