The mitochondrial genome

Question 1

What are the functions of the mitochondria?

Answer 1

ATP production

• Haem synthesis
• Neurotransmitter synthesis, e.g. glutamate
• Nucleotide synthesis

Question 2

Describe the structure of mitochondrial DNA

Answer 2

• Double stranded circular molecule (16.6kb) (15,000x smaller than chromosome 1)
• Consists of the heavy and light strand
• Multicopy genome (10-100,000 copies per cell)
• 37 genes
13 oxidative phosphorylation protein subunits
22 transfer RNAs
2 ribosomal RNAs
• No introns
• D-loop is a non coding region where replication and transcription are initiated
• Maternally inherited, no recombination

Question 3

Describe the proteins of oxidative phosphorylation

Answer 3

Mitochondria are required to produce 13 proteins of OXPHOS. OXPHOS consists of five enzyme complexes in the inner membrane of mitochondria. The first four complexes are the respiratory chain (RC) complexes (CI-CIV) and CV is the ATP synthase enzyme. Three of the RC complexes pump protons across the membrane generating electrochemical potential across the membrane. This potential is then utilized by CV to produce ATP. This is known as chemiosmosis and was proposed by Peter Mitchell in the 1960s for which he won a Nobel Prize.
Note that only 13 proteins required for OXPHOS are encoded by mtDNA. More than 100 are needed!

Question 4

What do the non-coding regions of DNA contain

Answer 4

Non-coding region (NCR) contains regulatory sequences for replication and transcription

Question 5

Where do replication and transcription start in mitochondrial DNA?

Answer 5

mtDNA replication starts in Origin of heavy strand (OH). Transcription starts at Heavy strand promoter (HSP) and Light strand promoter (LSP)

Question 6

How is mtDNA packaged?

What does Transcription factor A do?

Answer 6

• mtDNA is packaged into structures called nucleoids
One or two copies of mtDNA per nucleoid
Transcription factor A (TFAM) acts as histone protein
(Left) mtDNA (green) is located in nucleoids in tubular mitochondria (red). Nucleus is stained with DAPI (blue)
(Right) Transcription factor A also functions as a histone protein by packaging mtDNA

Question 7

How do population geneticists track migration of human populations?

Answer 7

• As mtDNA is maternally inherited different variants are restricted to different ethnic groups
• mtDNA does no recombine and mutations acquired over time subdivide the human population into discrete haplogroups

Question 8

What does mtDNA encode for?

What process’ must occur to produce these proteins?

Answer 8

•	mtDNA encodes 13 proteins of OXPHOS
o	But OXPHOS requires >100 proteins
•	To make the 13 OXPHOS proteins mtDNA must be: 
o	Replicated
o	Transcribed
o	Translated 

• All proteins involved in replication, transcription and translation of mtDNA are encoded by nuclear genes and imported into mitochondria
• In total >1000 mitochondrial proteins but only 13 made by mtDNA, all others made by nuclear genes!!

Question 9

What things are needed to replicate mitochondrial DNA?

Answer 9

• Mitochondrial DNA replication occurs continuously in cells. To replicate mtDNA you need:
• Polymerase gamma (POLG): mtDNA DNA polymerase
• TWINKLE: mtDNA helicase, unwinds DNA for replication
• Single stranded binding protein (SSBP): keep DNA unwound
• TFAM: packages and protects mtDNA
• In addition other proteins are needed e.g. enzymes important for making dTNTPs, the building blocks for DNA.

Question 10

Describe the structure of Mitochondrial DNA polymerase

Answer 10

• Polymerase gamma (Polg)
o Heterotrimer protein
 One catalytic subunit (POLgA)
 Two accessory subunits (POLgB)
• POLgA contain 3’ – 5’ exonuclease domain to proofread newly synthesized DNA
• POLgB enhances interactions with DNA template and increases activity and processivity of POLgA.

Question 11

Describe and name the mitochondrial helicase

Answer 11

• TWINKLE
o Hexamer – six TWINKLE subunits
• Unwinds double stranded mtDNA template to allow replication by Polg

Question 12

What is the function of Mitochondrial single stranded binding protein (mtSSBP)?

Answer 12

• Binds to single stranded DNA
o Protects against nucleases
o Prevents secondary structure formation
o Enhances mtDNA synthesis by stimulating TWINKLE helicase activity

Question 13

Describe the Strand displacement model of mtDNA replication

Answer 13

On image

Question 14

Describe the first phase in mtDNA replication

Answer 14

• Parental heavy strand displaced and coated with mtSSBP
• TWINKLE helicase unwinds mtDNA
• Mitochondrial RNA Polymerase (POLRMT) synthesizes RNA primer using light strand as template
• POLg uses RNA primer to replicate DNA at OH

Question 15

Describe the second phase in mtDNA replication

Answer 15

• Heavy strand replication passes OL
• Stem loop structure is formed preventing mtSSBP binding
• Mitochondrial RNA Polymerase (POLRMT) synthesizes RNA primer using heavy strand as template
• POLg uses RNA primer to replicate light strand DNA at OL

Question 16

Describe the third phase in mtDNA replication

Answer 16

Synthesis proceeds until both strands are fully replicated

Question 17

Describe mitochondrial diseases

Answer 17

•	Rare monogenic diseases
o	A disease is defined as rare when it affects less than 1:2000 individuals
•	Affect highly metabolic organs
o	Abundant in mitochondria
•	Can affect one or several organ systems. 
•	Start at any age
•	Wide disease spectrum e.g.
o	Hearing loss
o	Fatal cardiomyopathy in infancy
•	Genetically heterogenous
o	Mitochondrial or nuclear genes

Often these diseases are severe syndromes that affect multiple organs. Most typically these diseases affect the cns, muscles and the heart, but metabolic syndromes are also common. On the other end of the spectrum can also cause isolated symptoms such as hearing loss. However, in many patients, the clinical symptoms might be non-specific and mitochondrial disease is only one of several possible diagnoses.
How defects in mitochondria can cause such a diverse range of human diseases remains poorly understood.
Children may recover from one phenotype and later develop another — for example, in Pearson syndrome, the primary manifestation is exocrine pancreatic dysfunction and megaloblastic anaemia, and the survivors may later develop brain disease.
Typically, these disorders are progressive.
There are still no curative treatments

Question 18

Have a look at the mitochondrial DNA syndromes

Answer 18

On image

Question 19

What is the primary mitochondrial DNA mutations?

What are nuclear mutations?

Answer 19

Inherited for every offspring

On image

Question 20

Describe the purity of mtDNA

Answer 20

• Cells can contain pure (homoplasmy) or mixed (heteroplasmy) populations of mtDNA molecules.
• For mtDNA diseases usually minimum amount of mutation is required for diseases manifestation >80%.

Question 21

How can mtDNA mutations be identified?

Answer 21

mtDNA mutations can be identified by next-generation sequencing (NGS)
This graph shows that with NGS
the entire length of mtDNA is covered with several hundred short-reads. Therefore, method is highly reliable as no region is missing compared to nuclear genome.