Transcript Mitochondrial DNA

Mitochondrial Genome
Introduction
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membrane-bound organelle
Found only in eukaryotes
Contains a small DNA circle
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All eukaryotic cells either have mitochondria
or have nuclear genes that seem to have
been derived from mitochondria.
Each cell contains hundreds to thousands of
mitochondria.
Site of Krebs cycle and oxidative
phosphorylation (the electron transport
chain, or respiratory chain).
two membranes: outer and inner.
Folds of the inner membrane, where most of
oxidative phosphorylation occurs, are called
cristae.
Inside inner membrane = matrix (site of
Krebs cycle)
Between membranes = intermembrane
space
Mitochondrial DNA is in the matrix, inside
the inner membrane.
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Endosymbiont Theory
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endosymbiont theory: The mitochondria
(and also plant chloroplasts) originated
from an intracellular symbiosis between 2
different free-living organisms, which got
inside a primitive eukaryotic cell and
developed a mutually beneficial
relationship.
– originally proposed in 1883 by Andreas
Schimper, but extended by Lynn Margulis
in 1967.
– Also called symbiogenesis.
• Thought to have occurred about 1.5
billion years ago, at about the same
time that eukaryotes originated:
maybe the fundamental event in the
origin of eukaryotes.
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Mitochondrial ribosomal RNA genes and
other genes show that the original
organism was in the alpha-proteobacteria
family (similar to nitrogen-fixing bacteria)
More Endosymbiont Theory
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Evidence:
– mitochondria have their own DNA (circular)
– the inner membrane is more similar to prokaryotic membranes than to
eukaryotic. By the hypothesis, the inner membrane was the original prokaryotic
membrane and the outer membrane was from the primitive eukaryote that
swallowed it.
– mitochondria make their own ribosomes, which are of the prokaryotic 70S type,
not the eukaryotic 80S type.
– mitochondria are sensitive to many bacterial inhibitors that don’t affect the rest of
the eukaryotic cell, such as streptomycin, chloramphenicol, rifampicin.
– Also, inhibitors of eukaryotic protein synthesis don’t affect mitochondrial protein
synthesis.
– mitochondrial protein synthesis starts with N-formyl methionine, as in the bacteria
but unlike eukaryotes.
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Most of the original bacterial genes have migrated into the nucleus.
Mechanism unclear:
Eukaryotes that lack mitochondria generally have some mitochondrial
genes in their nucleus, evidence that their ancestors had mitochondria that
were lost during evolution.
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The origin of eukaryotes vs. origin of mitochondria are not clearly distinct events.
Phylogeny of Mitochondrial DNA
Endosymbiont Hypothesis
This is actually a secondary endosymbiosis: the largest cell is engulfing a photosynthetic eukaryote,
which already contains chloroplasts.
Mitochondrial Function
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Krebs cycle:
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Pyruvate, the product of glycolysis, is produced in the cytoplasm.
It is transported into the mitochondrial matrix (inside the inner membrane).
There, it is converted into acetyl CoA.
Fatty acids, from the breakdown of lipids, are also transported into the matrix and
converted to acetyl CoA.
– The Krebs cycle then converts acetyl CoA into carbon dioxide and high energy
electrons. The high energy electrons are carried by NADH and FADH2.
Electron Transport:
– The high energy electrons are removed from NADH and FADH2, and passed
through three protein complexes embedded in the inner membrane.
– Each complex uses some of the electrons’ energy to pump H+ ions out of the
matrix into the intermembrane space.
– The final protein complex gives the electrons to oxygen, converting it to water.
– The H+ ions come back into the matrix, down the concentration gradient, through
a fourth complex, ATP synthase (also called ATPase), which uses their energy to
generate ATP from ADP and inorganic phosphate.
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In brown fat, the synthesis of ATP is uncoupled from the flow of H+ ions
back into the matrix. The H+ ions flow through a protein called thermogenin,
and not through the ATPase. The energy is converted into heat: the
primary way we keep warm in cold weather.
Mitochondrial Function
Genome Structure
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The mitochondrial genome is a circle,
16.6 kb of DNA. A typical bacterial
genome is 2-4 Mbp.
The two strands are notably different
in base composition, leading to one
strand being “heavy” (the H strand)
and the other light (the L strand).
Both strands encode genes, although
more are on the H strand.
A short region (1121 bp), the D loop
(D = “displacement”), is a DNA triple
helix: there are 2 overlapping copies of
the H strand there.
The D loop is also the site where most
of replication and transcription is
controlled.
Genes are tightly packed, with almost
no non-coding DNA outside of the D
loop. In one case, two genes overlap:
they share 43 bp, using different
reading frames. Human mitochondrial
genes contain no introns, although
introns are found in the mitochondria
of other groups (plants, for instance).
Mitochondrial Genes
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Genes: total of 37. 22 transfer
RNAs, 2 ribosomal RNAs, 13
polypeptides.
Transfer RNA: only 60 of the 64
codons code for amino acids.
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Ribosomal RNA: 16S and 23S,
which are standard sizes for
bacterial rRNAs.
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Total of 22 tRNAs: 8 tRNAs cover all 4
3rd base positions with the same amino
acid, and the remaining 14 tRNAs each
cover two 3rd base positions (purines or
pyrimidines). Thus, all 60 codons are
covered.
Bacterial ribosomes don’t use 5S or
5.8S rRNAs.
polypeptides: all are components
of the electron transport chain.
Other components are encoded in
the nucleus and transported to the
mitochondria after translation.
Genetic Code
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The mitochondrial genetic code has drifted from the universal code:
there are so few polypeptides that changes in the code are tolerated.
Human mitochondrial code is different from other groups such as
plants or fungi.
Uses 2 of the 3 universal stop codons, but also uses 2 other codons
as stop codons. Also, UGA codes for tryptophan in the mitochondrial,
while it is a stop codon in the universal code. AUA gives methionine
in the mitochondria instead of isoleucine.
Replication
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Replication starts with the H strand.
The origin of replication for the H strand
(OriH) is in the D loop, and it is initiated by an
RNA primer generated from the L strand
transcript.
After the new H strand is about 2/3 complete,
the L strand origin of replication (OriL) is
uncovered. The L strand origin is on the old H
strand; it is “uncovered” when the old H strand
is displaced by the DNA polymerase
synthesizing the new H strand.
The L strand origin folds into a stem-loop
structure, which acts as a primer, and
replication of the L strand begins.
Replication can be said to be bidirectional but
asynchronous, unlike replication of nuclear
DNA, which proceeds in both directions
simultaneously.
Transcription and RNA Processing
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Both strands are transcribed as single RNA molecules
The D loop contains one promoter for each strand, and the entire strand is
transcribed.
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The RNA is then cut into individual RNAs for each gene. This involves at
least 43 separate cleavages.
Protein-coding genes are given poly-A tails.
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rRNA and tRNA molecules are modified as necessary.
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Transfer RNA molecules contain many modified bases
RNA editing also occurs: enzymes that convert specific C’s to U’s by
deamination
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No 5’ cap (which is found for nuclear transcripts)
No introns
Many mitochondrial transcripts have no 5’ UTR or 3’UTR (untranslated regions): translation
goes from the first base of the mRNA to the last base.
The first A of the poly A tail is the last base of the stop codon (UGA or UAA) for many
transcripts!
In plants, RNA editing in the mitochondria occurs at many sites
Mitochondrial RNA polymerase is coded in the nucleus, but it is not similar
to either eukaryotic or prokaryotic RNA polymerases.
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It seems to be a modified version of a bacteriophage RNA polymerase. It has just 1 subunit,
where typical eukaryotic RNA polymerases have 10-123 subunits and prokaryotic RNA
polymerases have 5 subunits
Transcription Map
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Heavy strand genes
are in the outer
circle; light strand
gens in the inner
circle.
There are 2 heavy
strand promoters:
H1 and H2, and a
light strand
promoter: LSP.
H1 makes a short
transcript for
ribosomal RNA only.
Black boxes are
protein-coding
genes; hatched
boxes are ribosomal
RNA genes; circles
are transfer RNA
genes.
RNA Editing
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RNA editing is a fairly rare form of RNA
processing, in which nucleotides are added,
removed, or altered in the RNA sequence after
transcription has occurred.
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RNA editing has been seen in all domains of
life, and in many different types of RNA,
including protein-coding messenger RNA.
One simple mechanism: an enzyme
deaminates a specific C in a mRNA,
converting it to U. In DNA this would be
repaired, but U is a legitimate RNA base.
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In apolioprotein, this change converts a CAA
(glutamine) codon into a UAA stop codon,
which allows translation of a much shorter
protein.
Another deamination, from adenine to inosine,
is common in mammals. Inosine acts like
guanine in translation and in base pairing.
Transporting Proteins into the Mitochondria
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Nuclear-coded proteins are transported across the
outer membrane by the translocase of the outer
membrane (TOM) complex, a multi-subunit integral
membrane protein.
If necessary, proteins are transported into or through
the inner membrane by the translocase of the inner
membrane (TIM) protein complexes, which has 2
independent components (TIM22 and TIM23), one for
inner membrane proteins and one for matrix proteins
Proteins destined for the matrix or inner membrane
have an N-terminal signal sequence:
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Very similar in function to signal sequence for endoplasmic
reticulum, except the mitochondrial proteins are completely
synthesized first, then transported. ER proteins are synthesized
directly into or through the membrane.
The signal sequence is an amphipathic alpha helix: charged on
one side and hydrophobic on the other side.
Recognized by components of TOM and TIM
Proteins destined for other locations have internal
signal sequences (not N-terminal)
Mitochondrial Protein Transport
Mitochondrial Genetics
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Maternal inheritance: Inherited
through the mother (egg) only. Allows
tracing female line back in time.
A few sperm mitochondria enter the
egg, but they are degraded and lost.
Mutation rate in mtDNA is very high:
10 times the nuclear rate.
mtDNA is associated with the inner
membrane, the site of oxidative
phosphorylation.
Oxidative phosphorylation generates
large amounts of “reactive oxygen
species” (peroxide and superoxide),
which are quite mutagenic.
Heteroplasmy
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Sometimes an individual has more than one
kind of mitochondria. This is called
heteroplasmy.
Since mitochondria are divided randomly
during cell division, different cells get
different proportions of the two types.
If one mitochondrial type is mutant and the
other is normal, severity of symptoms will
vary in different tissues depending on the
proportions of the two types.
Inheritance: During oogenesis (egg
formation), random segregation of the two
types can lead to some offspring inheriting a
mitochondrial disease while other offspring
are normal.
– No simple segregation ratio: a sign of a
mitochondrial disease
– Severity of symptoms varies between
offspring
– Oocytes contain only about 10
mitochondria
Genetic Diseases
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in general: malfunctions of respiratory chain affect high metabolism tissues the most:
nervous system, muscles, kidney, liver.
Heteroplasmy is very common, meaning that symptoms vary widely and affect different
tissues at different rates
Mutations in many different genes can produce similar diseases
Leber's Hereditary Optic Neuropathy
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Progressive loss of vision, from central to
peripheral, usually beginning in 20's.
Eyes can be affected several months apart, or
simultaneously.
About 85% are male (no good reason why).
Recurrence risk for siblings around 20%
(heteroplasmy); many spontaneous cases.
Due to death of optic nerve fibers.
Most due to change in conserved Arg to His in
NADH dehydrogenase, but 18 total mutations
known, all missense in respiratory chain.
MERRF
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Myoclonic epilepsy and ragged red
fiber disease (MERRF). CNS
symptoms: epilepsy, deafness,
dementia. Muscle twitching and
weakness. Skeletal and heart muscles
appear abnormal, mitochondria appear
abnormal. Multiple enzyme defects in
respiratory chain.
Ragged red fibers are clumps of
abnormal mitochondria that
accumulate in the muscle fibers that
stain red using a standard histological
stain.
Most (80%) MERRF cases are due to
A --> G mutation in lysine tRNA
(mutation A8344G). Easy to assay
for; CviJI restriction site is altered.
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Some cases involve other tRNA genes
Lots of variation in inheritance of
disease, due to heteroplasmy.
Good correlation between % mutant
mitochondria and disease severity.
Kearns-Sayre Syndrome
• Symptoms: paralysis of the muscles, droopy eyelids, retinal
degeneration, cardiac muscle problems, seizures, many other
symptoms irregularly.
• Cause: Large deletions of mtDNA in the protein coding gene
region. This leads to a failure of oxidative phosphorylation, thus
low energy production in the cell.
• Heteroplasmy necessary for survival. Mostly spontaneous--rarely
passed to offspring. Many variants.
Mitochondria, Free Radicals, and Aging
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One theory of aging: DNA and other biological
molecules are damaged by oxidation. Damage is
caused by reactive oxygen species (ROS):
molecules containing oxygen that act as strong
oxidizing agents.
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Damage: crosslinking of DNA, oxidation of lipids and
proteins
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free radicals: molecules that have an unpaired electron.
Examples: superoxide: O2- and nitric oxide (NO). They attack
other molecules by pulling off an electron (oxidation), creating
new free radicals.
Also bad: peroxide
Mitochondria that have too much oxidative damage induce
apoptosis (programmed cell death)
ROS are created by ionizing radiation and energetic
chemical reactions (cigarette smoking, for example).
Also created in the mitochondria as a byproduct of
the electron transport chain. Usually, the last step
involves reducing an O2 molecule to water by
donating a pair of electrons. However, sometimes
only a single electron gets donated, producing
superoxide instead of water.
Dealing With ROS
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We have enzyme systems
to remove superoxide,
peroxide, and other ROS:
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Superoxide dismutase
converts superoxide to
peroxide
Catalase converts peroxide to
water
Also: anti-oxidants (which
have extra electrons to
donate to the free radicals)
remove ROS
More Aging
• The theory: mitochondrial
DNA mutated by ROS
produces defective electron
transport proteins, which in
turn generate more free
radicals: positive feedback.
– ROS-stressed mitochondria
induce apoptosis
– The free radicals oxidize lipids
and proteins in the cells
– Causes wrinkled skin, cancer,
arterial plaque formation, many
other chronic age-related
diseases
Three Parent In Vitro Fertilization
• The idea is to use the nucleus from one woman and the egg
cytoplasm (especially the mitochondria) from another woman.
– This allows woman with defective mitochondrial DNA to produce healthy children.
• Procedure: remove the nucleus from a healthy egg, then transfer in
the nucleus from an egg with bad mitochondria. Then, fertilize in
vitro with a sperm nucleus.
• Has been performed in Great Britain, but currently banned in the
US. It is a form of gene therapy that affects future generations; such
procedures are viewed cautiously.