Mitochondrial genome

• Mitochondria are dynamic organelles that are present
in almost all eukaryotic cells and play a crucial role in
several cellular pathways. Their most recognizable role
is providing the cell with energy in the form of ATP via
OxPhos. However, many other functions have been
assigned to mitochondria, including the integration of
metabolic pathways (such as the biosyntheses of
heme, iron–sulfur clusters, and nucleotides), apoptosis,
and reactive oxidative species (ROS) signaling

• The endosymbiotic theory proposes that mitochondria originated
as free-living Alphaproteobacteria that were internalized by a pre-
eukaryotic host cell, leading to the formation of the modern
eukaryotic cell. In the course of evolution, the genome of the
original alphaproteobacterial symbiont has undergone extensive
reduction. The majority of its genes have either been lost, owing to
redundancy, or transferred to the host nuclear genome.
Furthermore, mitochondria have lost autonomy over their genome
maintenance and expression to the host cell. Nonetheless, in
almost all cases, eukaryotic mitochondria retain a minimal genome,
of variable size and gene content, that is present in many copies
within their matrix.

• Human mitochondrial DNA (mtDNA) is a circular molecule of 16.5
kb which encodes a small subset of the structural polypeptide
components required for OxPhos. These mRNAs aretranscribed and
then translated within the mitochondrial matrix by a dedicated,
unique, and highly specialized machinery. The RNA components of
the mitochondrial gene expression system, two mitochondrial
ribosomal RNAs (mt-rRNAs) and 22 mt-tRNAs, are also encoded by
mtDNA, whereas all other protein components are encoded by
nuclear genes and imported into mitochondria from the cytosol

• 250–300 nucleus-encoded proteins are dedicated
to serve mitochondrial gene expression. This
includes RNA polymerase and transcription
factors, endonucleases for RNA precursor
processing, aminoacyl-tRNA synthetases, RNA-
modifying enzymes, the structural components
and biogenesis factors for the mitochondrial
ribosome, translation factors, and other auxiliary
factors

• Map of the human mitochondrial genome (16 569
bp). The outer circle represents the H-strand,
containing the majority of the genes; the inner circle
represents the L-strand. The D-loop is shown as a
three-stranded structure. The origins of H-strand (OH)
and L-strand (OL) replication and the direction of DNA
synthesis are indicated by long bent arrows; the
initiation of transcription sites (ITL, ITH1, ITH2) and the
direction of RNA synthesis are denoted by short bent
arrows. The binding site for the mitochondrial
transcription terminator (mtTERM) is indicated. The
22 tRNA genes are depicted by dots and the single
letter code of the amino acid (isoacceptors
for serine and leucine are distinguished by
their codon sequence). The genes coding for the two
rRNA species (12S and 16S) and the 13 protein coding
genes are depicted by shaded
boxes. ND, CO and ATPase refer to genes coding for
subunits of NADH:ubiquinone oxidoreductase,
ferrocytochrome c:oxygen oxidoreductase
(cytochrome c oxidase) and F1F0-ATP synthase,
respectively, whereas Cyt b encodes
apocytochrome b of
ubiquinol:ferricytochrome c oxidoreductase.

Basic Features
• The strands of the DNA duplex can be distinguished on the basis of
G+T base composition which results in different buoyant densities of each
strand (‘heavy’ and ‘light’) in denaturing caesium chloride gradients
• Most information is encoded on the heavy (H) strand, with genes for two
rRNAs, 14 tRNAs, and 12 polypeptides.
• The light (L) strand codes for eight tRNAs and a single polypeptide.
• All 13 protein products are constituents of the enzyme complexes of
the oxidative phosphorylation system
• The genes lack introns and, except for one regulatory region, intergenetic
sequences are absent or limited to a few bases. Both rRNA and tRNA
molecules are unusually small
• Some of the protein genes are overlapping and, in many cases, part of the
termination codons are not encoded but are generated post-
transcriptionally by polyadenylation of the mRNAs

Unique Codons
• mitochondrial protein sequences revealed deviations from
the standard genetic code and later even variations in codon
usage were found in mitochondria from different species
• In mtDNA of most phylogenetic groups, TGA is used as
a tryptophan codon, rather than as a termination codon.
• AGR (R=A, G) specifies a stop in mtDNA of vertebrates, codes
for serine in mtDNA of echinoderms and codes for arginine
in mtDNA of yeast, as in the standard genetic code.

mtDNA Inheritance
• • mtDNA is inherited entirely from the mother
• • Sperm carries the father's mtDNA in its tail, which is lost
• during fertilization
• • mtDNA inheritance is non-Mendelian, because
• Mendelian inheritance presumes that half the genetic
• material of an embryo derives from each parent
• • mtDNA passes unchanged from mother to offspring by
• this mechanism

Characteristics of mtDNA
• The two strands of mtDNA have significantly different
compositions from nuclear DNA and from one another
- The heavy (H) strand is rich in purines (adenine and
guanine)
- The light (L) strand is rich in pyrimidines (thymine and cytosine)
• mtDNA is highly conserved, so it is useful for
phylogenetic study
• 80% of mtDNA encodes for functional mitochondrial
proteins, and therefore most mtDNA mutations lead to
functional anomalies
• Mutations in nuclear DNA may also have a wide array of
effects on mtDNA replication
• mtDNA is devoid of introns
mtDNA contains only a few non-coding
intergenic regions
• The size and number of mtDNA
- Human mtDNA is 16,569 bp in length
- Human cells typically contain
thousands of copies of
mtDNA, several copies per
mitochondrion

mtDNA genes
mtDNA encodes for 37 genes
• There are 13 peptide coding genes, including the following
peptides
• Transcription factor A
• The mtRNA processing ribonuclease P
• The transcription termination factor
• The mitochondrial peptides are synthesized on
mitochondrial ribosome
• The heavy strand encodes the 2 rRNAs, 12 polypeptides, and
14 tRNAs
• The light strand encodes 1 polypeptide, and 8 tRNAs

mtDNA expression
- Transcription initiation sites of mtDNA
• The promoters of Hand L strands (termed PH and
PL) are both located in the D-Ioop region and 150
bp apart (seemtDNA replication at the end of this
section) (Figure 26)
• Heavy-strand transcription starts at nucleotide
561
• Light-strand transcription starts at nucleotide 407

Transcription of mtDNAstarts from the promoters in the D-Ioop
region and continues in opposing directions for the two
strands around the circle to generate large multigenic
transcripts
• Transcription initiation in mitochondria involves three types
of proteins
• The mtRNApolymerase (POLRMT)
• Mitochondrial transcription factorA (TFAM)
• Mitochondrial transcription factors BI and B2 (TFBIM, TFB2M)
• POLRMT, TFAM,and TFBIMor TFB2M assemble at the
mitochondrial promoters and begin transcription

Promoters of mitochondrial gene expression
• Heavy strand I (HI) promoters initiate
transcription of the entire heavy strand
• Heavy strand 2 (H2) promoters initiate
transcription of the two mitochondrialrRNAs
• Light strand (L) promoter initiates transcripts
of the entire light strand

Mitochondrial mRNA
- Mitochondrial mRNAs are small molecules
- Full-length transcripts are cut into functional
tRNA,
rRNA, and mRNAmolecules
- Mitochondrial mRNAlacks a 5' cap structure
- Mitochondrial mRNAslack both a 5' and a 3' UTR
• - The first codon specifies N-formylmethionine
and is located at or very near the 5' end

Regulation of mtDNAexpression
- mtDNA expressiondepends on a large number of
proteins encoded by nuclearDNA
- The regulatory proteins are synthesized in the
cytosol and enter the mitochondria via
specialized pores
- mtDNA replication is regulated by only one
regulatory region controlling both the heavy and
the light strands

Post-transcriptional modification of mitochondrial
mRNA
- Mitochondrial mRNAs are processed by mitochondrial
ribonuclease (mtRNase)cleavage of the transcript
- The light strand may produce either short transcripts,
which serve as primers for mtDNAreplication, or a long
transcript for peptide and tRNAproduction
- The productionof primer occurs by processing of light-
strand transcripts with the mtRNAseP
- The Hand L strand mRNAmolecules are polyadenylated
by a mitochondrial poly(A) polymerase, imported from
the cytosol

Mitochondrial mRNA translation
- The protein components necessary for mitochondrial translation,
including ribosomal proteins, tRNA synthetases, ribonucleases,
initiation, and elongation factors, are all encoded by nuclear
genes.
- Mitochondrial protein synthesis and DNA replication are thus
under nuclear regulatory control
- Mitochondrial translation is bacteria-like both in its sensitivity to
antibiotics that act on the ribosome, and in its use of N-
formylmethionyl-tRNA for initiation
- Mitochondrial ribosomes are smaller than those found in the
cytosol, and have a sedimentation coefficient of 55 S instead of
the denser 80 S sedimentation coefficient for cytosolic ribosomes
or 70 S coefficient for bacterial ribosomes

mtDNA Replication
• mtDNA replication is an asynchronou s process, which
begins at the origin of the H strand mtDNA replication
is controlled by chromosomes in the nucleus based on
how many mitochondria the particular cell needs at
that time
- When the replication apparatu s meets the origin of
the L strand, it is forced into a single-strand
configuration by the extending daughter H strand, and
L-strand replication begins at this point RNA derived
from the L-strand promoter serves as a primer for H-
strand DNA replication.

The D-Ioop (displacement loop) is a I I23-ba se stretch of DNA, often triple-
stranded, which contains sites for DNA-binding protein s that control
mtDNA replication and transcription
- The D-Ioop contains the promote rs for both the H- and L-strand transcripts
- mtDNA replication causes the D-Ioop to move along the heavy strand as
mtDNA polymerase-y produces a complimentary replica strand
• Heavy-strand DNA replication begins at the D-Ioop and proceed s in a 5'-3'
direction until returning to the origin of replication
• DNA polymerase y begins in the reverse direction to produce a
complimentary replica of the lightstrand when replication of the heavy
strand reaches the light strand replication origin (OL)
• Two identical double-strand mtDNA molecules are the result of this process
• When mitochondria have enough copies of mtDNA, sufficient mitochondrial
proteins, and adequate surface area, a nuclear protein may permit the
mitochondrion to divide by fission into two daughter mitochondria

• - mtDNA is susceptible to insult by all the same processes that
damage nuclear DNA
• - mtDNA is especially susceptible to insult by reactive oxygen
species, which are prevalent in mitochondria
• • Because mtDNA is not bound to histones, it is exposed to damage
caused by free oxygen radicals produced by electron transfer during
oxidative phosphorylation of the respiratory chain
• • mtDNA also undergoes the same types of mutation as nuclear
DNA including spontaneous modifications and replication errors
• mtDNA damage

mtDNA mutations
- The rate of mutation in mtDNA is calculated to be about 10 times greater
than that of nuclear DNA
- The mtDNA mutations may be either acquired or inherited
- Several different mutations of mtDNA may present clinically as the same
disease
- Large deletions and duplications of mtDNA increase with age
• This may account for some aging processes in oxygen-dependent organs,
such as brain, kidney, muscle, and heart
• Mutant electron transfer proteins may release more oxygen-free radicals
into the mitochondrial matrix, accelerating the aging process in some
cases of Alzheimer's and coronary artery disease
- There are hypervariable segments (HVI and HV2) located at base 57-372 and
base 16,024-16,383, respectively. The rate of mutation in these regions is
significantly higher than in the rest of mtDNA

mtDNA repair
- mtDNA does not code for any DNA repair proteins
- Proteins from the cytosol under nuclear control enter the
mitochondrion through specialized membrane pores
- Recent evidence has suggested that mitochondria have
enzymes to proofread mtDNA and fix mutations owing to free
radicals
- Evidence for nucleotide excision repair, direct damage
reversal, mismatch repair, and recombinational repair
mechanisms have also been found in mitochondria
- As with nuclear DNA repair, the ability of mitochondria to
repair DNA damage declines with age

Mitochondrial Disease
• Mitochondrial diseases result from failures of the mitochondrial specialized compartments
for oxidative phosphorylation, which are present in every cell of the body except red blood
cells
- About one in 4000 children in the United States will develop a mitochondrial disease by the
age of 10 years
- 1000-4000 children per year in the United Sates are born with some type of congenital
mitochondrial disease
- Mitochondrial diseases may either be observable at birth, or symptoms may not be seen until
late adulthood
- Heteroplasmy refers to a phenomenon in which the number of mutant versus wild-type
mitochondria varies from cell to cell and from tissue to tissue When a tissue reaches a
certain ratio of mutant to wild-type mitochondria, a disease becomes manifest
- Mitochondrial disease may be caused either by mtDNA mutations (acquired or inherited) or
by mutations in nuclear DNA coding for mitochondrial components
- Types of mutations
• Homoplasmic: similar distribution of mtDNA mutation in all tissues
• Heteroplasmic: variable distribution of mtDNA mutation in different cells or tissues

• Typical symptoms of mitochondrial disease include
- Loss of muscle coordination, muscle weakness Neurologic
problems, seizures
Visual and/or hearing problems Developmental delays,
learning disabilities Heart, liver, or kidney disease
Gastrointestinal disorders and severe constipation Diabetes
Increased risk of infection Thyroid and/or adrenal
dysfunction Autonomic dysfunction Neuropsychologic
changes characterized by confusion, disorientation, and
memory loss

The diagnosis of mitochondrial disease is problematic
There is no reliable and consistent means of diagnosis
Evaluating the patient's family history is essential
- Diagnosis may require one of the few physicians who
specialize in mitochondrial disease
- Diagnosis can be made by blood DNA testing and/or
muscle biopsy but neither of these tests is completely
reliable

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