Molecular biology of mitochondria

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Molecular Biology 325 2007
Molecular biology of mitochondria
Mitochondria are the main site of ATP synthesis
in eukaryote cells and as such are vital for the
health and survival of the cell They are also
one of the sites at which apoptosis is mediated
These lectures will explore the molecular
genetics of mitochondria, how they are made, the
structure of their genome, how they evolved , and
how mitochondrial gene expression is controlled.
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Mitochondrial molecular genetics 1 focus on
mitochondria brief overview of their function
and structure mtDNA structure and
replication - animals - yeast - plants
inheritance of mitochondria - petite mutants of
yeast biogenesis of mitochondria by fission
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MITOCHONDRIA essential for cell life - ATP
synthesis - many metabolic intermediates
essential for cell death - unprogrammed
death necrosis ( eg, due to loss of energy
status) - programmed cell death (apoptosis
- controlled cell destruction)
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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
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Overview of aerobic respiration
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Outline of Tricarboxylic Acid Cycle
3-Carbons
One pyruvate molecule is completely oxidised to
CO2
CO2
4-Carbons
6-Carbons
NADH
NADH CO2
The NADH and FADH produced are oxidised by the
respiratory electron transport chain
FADH
NADH CO2
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Four large, multi-subunit protein complexes -
complex I is a NADH-ubiquinone 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
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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
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Mitochondria also have their own ribosomes and
tRNA 22 tRNAs rRNAs (16S and 12S)
The ribosomes can actually be visualized in some
mitochondria. In these figures, they are seen in
the matrix as small dark bodies. DNA can also be
visualized in mitochondria. The DNA is circular
and resembles that of a bacterium in its basic
structure.
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To visualize the structure of mitochondrial DNA,
we have to extract the DNA and float it on a
water surface. Then, it can be picked up by a
plastic coated grid, and examined in the electron
microscope. Mitochondrial circular DNA is shown
in the figure.
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Mitochondrial Inheritance Yeast has been used
extensively to study mitochondrial
inheritance. There is a Yeast strain, called
"Petite" that have structurally abnormal
mitochondria that are incapable of oxidative
phosphorylation.  These mitochondria have lost
some or all of their DNA. Genetic crosses
between petite and wt strains showed that
inheritance of this trait did not segregate with
any of the nuclear chromosomes.
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Mitochondrial Inheritance
Mitochondrial inheritance from yeast is
biparental, and both parent cells contribute to
the daughter cells when the haploid cells fuse. 
After meiosis and mitosis, there is random
distribution of mitochondria to daughter cells. 
If the fusion is with yeast that are petite and
yeast that are not, a certain percentage of the
daughter cells will be "petite".  
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Mitochondrial Inheritance in Yeast
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Mitochondrial Inheritance
This led to the suggestion that some genetic
element existed in the cytoplasm and was
inherited in a different manner from nuclear
genes. This is called non-Mendelian
inheritance or cytoplasmic inheritance.
In yeast and animals, this indicated inheritance
of mitochondrial genes in plants it also
includes inheritance of chloroplast genes
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Mitochondrial replication
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Mitochondrial replication
Mitochondria replicate much like bacterial cells.
When they get too large, they undergo fission.
This involves a furrowing of the inner and then
the outer membrane as if someone was pinching the
mitochondrion. Then the two daughter mitochondria
split. Of course, the mitochondria must first
replicate their DNA. An electron micrograph
depicting the furrowing process is shown in these
figures.
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Sometimes new mitochondria are synthesized in
centres that are rich in proteins and
polyribosomes needed for their synthesis. The
electron micrograph in the following figure shows
such a centre. It appears that the cluster of
mitochondria are sitting in a matrix of proteins
and other materials needed for their production.
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Certain mitochondrial proteins are needed before
the mitochondria can divide. This has been shown
in a study by Sorgo and Yaffe, J Cell Bio. 126
1361-1373, 1994. They showed the result of the
removal of an outer membrane protein from
mitochondria called MDM10. This figure shows the
results. The mitochondria are able to take in
components and produce membranes and matrix
enzymes. However, fission is not allowed and the
result is a giant mitochondrion.
giant mitochondrion
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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.
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Yeast mitochondrial chromosome
yeast mtDNA
human mtDNA
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Human DNA 16,569 bp no non-coding DNA
no introns polycistronic replication followed
by splicing to form messages. Yeast mtDNA
68-75 kb, similar in structure to bacterial
genome contains introns and non-regions between
genes. Same proteins made as in animals genes
transcribed separately
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Despite having their own genome, most
mitochondrial proteins are encoded in the
nucleus, made in the cytosol and imported into
the mitochondria
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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.
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Plant mtDNA chromosome size is much bigger but
varies dramatically between species (200-2000
kb) arranged as different size circles,
sometimes with plasmids. The plant mtDNA
contains chloroplast sequences, indicating
exchange of genetic information between
organelles in plants. Much of the plant mtDNA
is non-coding, but coding regions are larger than
animals and fungi. Number of proteins
synthesised not known definitely but more than in
animals and yeast (probably about 50)
Plant mitochondria have specialised functions
in leaves they participate in photorespiration
sites of vitamin synthesis (vit C, folic acid,
biotin)
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maize mitochondrial genome
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In plants, respiration and photosynthesis operate
simultaneously in the light
DAY
NIGHT
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Chloroplasts are the site of photosynthesis and
belong to the plastid family of organelles - they
develop from proplastids in the light
proplastid
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Rice mitochondrial and chloroplast genomes Plant
mitochondria contain chloroplast genes -
suggesting that genetic transfer occurs between
the two organelles
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Mitochondrial DNA of animals and fungi uses a
different genetic code than the universal code
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RNA processing in mitochondria
Plant mitochondria edit their RNA transcripts.
This was first noticed when comparing cDNA
sequences with genomic DNA sequences. The most
common change is to replace C with U, although in
some instances other changes can occur. Matrix
enzymes are thought to be responsible for this,
but the reason for the editing is not
known. Most of the DNA in plant mitochondria is
non-coding, only some of which is transcribed.
RNA editing occurs even in non-coding regions
such as introns.
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Evolution of mitochondria
Mitochondria are generally thought to have
evolved endosymbiotically when an anaerobic
prokaryotic cell engulfed an aerobic bacterium
and formed a stable symbiosis. Loss of most of
the aerobes genome to the nucleus of the host
allowed the latter to control the former.
This is supported by gene sequence analysis which
shows remarkable homology between bacteria and
mitochondrial genes.