The mitochondrial genome

Question 1

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

The folds of the inner mitochondrial membrane

Answer 2

cristae which increase surface area for oxidative phosphorylation

Question 3

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

Functions of Mitochondria

Answer 4

Mainly generation of ATP, but also:

• purine intermediates required for DNA/nucleotide synthesis
• haem synthesis
• neurotransmitter synthesis (e.g. glutamate)
• important for apoptotic and necrotic cell death
• important in inflammation;

Question 5

Features of Mitochondrial DNA (mtDNA)

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
• D loop where replication and transcription is initiated

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

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
• ATP synthase is the (5th) CV complex

Question 8

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 (ATP synthase) 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 and contains regulatory sequences for this

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

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 as in nuclear DNA)

Question 16

mtDNA haplogroups

Answer 16

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

Question 17

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

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

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

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

mtDNA Single Stranded Binding Protein (mtSSBP)

Answer 22

Binds single stranded DNA to:

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

Question 23

Which model represents mtDNA replication?

Answer 23

Strand displacement model

Question 24

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 Topoisomeras (Top 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

Mitochondrial diseases

Answer 27

• rare monogenic diseases
• oxidative phosphorylation disorders 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

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

LHON (Leber’s Hereditary Optic Neuropathy)

Answer 29

• Mitochondrial Inheritance - homoplasmic mutation (all children affected)
• degeneration of retinal ganglion cells and their axons
• Acute loss of vision
• affect males more than females

Question 30

Mitochondrial disease inheritance

Answer 30

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 31

Heteroplasmy

Answer 31

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 32

Heteroplasmy inheritance

Answer 32

inheritance of heteroplasmy (mutation load) is very random

Question 33

How can mtDNA mutations be identified?

Answer 33

by next generation sequencing (NGS)

Question 34

Treatments of mitochondrial diseases

Answer 34

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

Question 35

mtDNA mutations and disease complexity

Answer 35

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.

Question 36

main steps of mitochondrial replication as shown by the strand displacement model

Answer 36

1. replication of heavy strand
2. replication of light strand
3. replication of both strands completed
4. segregation of daughter molecules