The mitochondrial genome

Question 1

Describe the mitochondrial structure.

Answer 1

Double membrane consisting of outer and inner membrane

Separating outer and inner membrane is intermembrane space

Within inner membrane is the mitochondrial matrix which contains mitochondrial DNA and ribosomes distinct from cytosolic ribosomes

Question 2

What are The folds of the inner mitochondrial membrane?

Answer 2

cristae which increase surface area for oxidative phosphorylation

Question 3

What is the Origin of mitochondria?

Answer 3

bacteria

-single event where primitive eukaryote engulfed a bacterial cell, giving rise to possibility for complexity in organisms (e.g. multicellularity)

Question 4

What are the Functions of Mitochondria?

Answer 4

Mainly generation of ATP, but also:

• purine intermediates required for DNA/nucleotide synthesis
• calcium homeostasis
• haem synthesis
• neurotransmitter synthesis (e.g. glutamate)
• important for apoptotic and necrotic cell death
• production of reactive oxygen species (can be damaging, but also required for signalling)
• important in inflammation; mitochondrial anti-viral signalling complex (MAVS) on outside of mitochondria is essential for innate immunity

Question 5

Gives some features of Mitochondrial DNA.

Answer 5

• Double stranded circular DNA (heavy and light strand)
• Multi-copy genome, unlike diploid cells which just have two copies of each gene in the nucleus
• No introns
• 16kp

Question 6

How many genes are in mtDNA?

Answer 6

37 genes
>13 encode oxidative phosphorylation protein subunits (OXPHOS proteins)
>22 encode tRNAs
>2 encode rRNAs

Question 7

What are OXPHOS proteins?

Answer 7

OXPHOS (oxidative phosphorylation) consists of 5 enzyme complexes (CI-CV) in the inner membrane of the mitochondria:
>CI-CV complexes are respiratory chain complexes
>ATPsynthase is the CV complex

Question 8

What is the Function of respiratory chain complexes?

Answer 8

Three of the respiratory chain complexes pump protons across the membrane generating an electrochemical potential across the membrane

This potential is utilised by CV (ATPsynthase) in chemiosmosis to produce ATP

Question 9

What is a D-loop in mtDNA?

Answer 9

non-coding region where replication and transcription are initiated

Question 10

Where is mitochondria inherited from?

Answer 10

thought to be strictly maternally inherited and there is no recombination

Question 11

What does the mitochondrial genome encode?

Answer 11

13 proteins of oxidative phosphorylation (OXPHOS proteins)

*note only 13 proteins for OXPHOS encoded by mtDNA, but OXPHOS requires more than 100 proteins, meaning rest are made by nuclear genes and imported into mitochondria

Question 12

What does the non-coding region (D-loop) of mtDNA contain?

Answer 12

regulatory sequences important for mitochondrial replication and transcription

Question 13

Where does mtDNA replication and transcription start from?

Answer 13

mtDNA replication starts in the D-loop of the non-coding region in the Origin of heavy strand

mtDNA transcription starts at Heavy strand promoter (HSP) and Light strand promoter (LSP)

Question 14

What is the mtDNA packaged into?

Answer 14

little structures called nucleoids by transcriptional factor A (TFAM) in order to prevent it from replicating or being damaged by reactive oxygen species
-one or two copies of mtDNA per nucleoid

Question 15

What are the Exceptions in mtDNA to the “universal” genetic code?

Answer 15

· Genetic code in vertebrate mitochondria:
>AUA and AUG code for methionine (AUA codes for isoleucine in nuclear DNA)
>UGA codes for tryptophan (stop codon in nuclear DNA)
>AGA and AGG are stop codons (not arginine)

Question 16

What are the mtDNA haplogroups?

Answer 16

no recombination in mtDNA and therefore mutations acquired over time subdivide the human population into discrete haplotypes

Question 17

What are the Requirements of mtDNA to produce OXPHOS proteins?

Answer 17

To make the 13 OXPHOS proteins, mtDNA must be:

• replicated
• transcribed
• translated

*all proteins involved in replication, transcription and translation of mtDNA are encoded by nuclear genes and imported into mitochondria

Question 18

Describe the mtDNA replication machinery..

Answer 18

· Polymerase gamma (PolG): mtDNA DNA Polymerase

· TWINKLE: mtDNA helicase, unwinds DNA for replication

· Single stranded binding protein (SSBP): keeps DNA unwound

· TFAM: packages and protects mtDNA

Question 19

What is mtDNA Polymerase?

Answer 19

Polymerase Gamma:

-heterotrimeric protein

Question 20

What are the subunits of polymerase gamma?

Answer 20

one catalytic subunit (polymerase gamma A)
-contains 3’-5’ exonuclease domain to proofread newly synthesized DNA

two accessory subunits (polymerase gamma beta)
-enhance interactions with DNA template and increase activity and processivity of polymerase gamma A

Question 21

What is mtDNA TWINKLE?

Answer 21

TWINKLE is mtDNA helicase with 6 different subunits (hexamer)
- unwinds double stranded mtDNA template to allow replication by polymerase gamma

Question 22

What is the mtDNA Single Stranded Binding Protein?

Answer 22

Binds single stranded DNA to:

• protect against nucleases
• prevents secondary structure formation
• enhances mtDNA synthesis by stimulating helicase activity

Question 23

Which model represents mtDNA replication?

Answer 23

The strand displacement model

Question 24

What is the Strand displacement model of mtDNA replication?

Answer 24

Step 1:

• parental heavy strand displaced and coated with mtSSBP
• TWINKLE helicase unwinds mtDNA
• mitochondrial RNA polymerase (POLRMT) synthesises RNA primer using light strand as template
• polymerase gamma uses RNA primer to replicate DNA at origin of heavy strand

Step 2:

• once heavy strand replication passes origin of light chain, origin of light chain forms a stemmed loop structure
• this prevents mtSSBP from binding anymore and frees up space for mitochondrial RNA polymerase (POLRMT) to synthesise RNA primer using heavy strand as a template
• polymerase gamma then uses RNA primer to replicate light strand DNA at origin of light chain

Step 3:

• synthesis proceeds until both strands are fully replicated
• two strands are still connected to each other and need to be separated

Step 4:

-daughter molecules are segregated by a protein called Topoisomerase 3a

Question 25

What happens if Topoisomerase 3a is deleted?

Answer 25

daughter mtDNA molecules cannot be segregated, resulting in a catenated molecule

Question 26

Why are mtDNA strands named “heavy” and “light”?

Answer 26

because heavy strands contain more purines which are heavier than pyrimidines

Question 27

Give some effects of mitochondrial diseases.

Answer 27

• rare monogenic diseases
• affect highly metabolic organs (because these are highly abundant in mitochondria)
• can affect one or several organ systems
• both genders affected (can start at any age)
• heterogenous (mitochondria or nuclear genes)

Wide disease spectrum e.g.:

• hearing loss
• fatal cardiomyopathy in infancy

Question 28

What is Leigh Syndrome?

Answer 28

most common mitochondrial disease presentation:
-neurological disorder characterised by progressive loss of mental and movement abilities (psychomotor regression) and typically results in death within two to three years, usually due to respiratory failure

Question 29

What is LHON (Leber’s Hereditary Optic Neuropathy)?

Answer 29

• Mitochondrial Inheritance
• degeneration of retinal ganglion cells and their axons
• Acute loss of vision
• affect males more than females

Question 30

What is KSS (Kearns-Sayre syndrome)?

Answer 30

-Large deletion of mtDNA

Question 31

Give some other mtDNA syndromes.

Answer 31

MELAS
-mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes

MERRF
-myoclonus epilepsy red ragged fibres

NARP
-neurogenic muscle weakness ataxia retinitis pigmentosa

Question 32

Describe the Mitochondrial disease inheritance.

Answer 32

Because of the maternal inheritance of mitochondria, for mitochondrial genome mutations, you will see maternal inheritance. But because nuclear gene mutations also cause mitochondrial diseases, all kinds of inheritance patterns are possible (e.g. Autosomal dominant, Autosomal recessive, X-linked and even de novo mutations)

Question 33

What is Heteroplasmy?

Answer 33

Presence of both normal and mutated mtDNA within a cell, resulting in variable expression in mitochondrial inherited disease

Most mtDNA show heteroplasmy

Heteroplasmy levels determine disease manifestation, as variant has to be present in high loads (>80%) before it can actually cause any symptoms

Question 34

Describe the inheritance of heteroplasmy.

Answer 34

inheritance of heteroplasmy (mutation load) is very random

Question 35

How can mtDNA mutations be identified?

Answer 35

By next generation sequencing (NGS)

Question 36

What are the Treatments of mitochondrial diseases.

Answer 36

no therapies and generally all mitochondrial diseases are incurable except for a couple

Question 37

Describe the mtDNA mutations and disease complexity.

Answer 37

mtDNA mutations tend to involve very severe monogenic disease, however common variants in mtDNA can contribute to development of complex diseases e.g. diabetes, Parkinson’s disease.