The mitochondrial genome Flashcards
Mitochondrial Structure
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
The folds of the inner mitochondrial membrane
cristae which increase surface area for oxidative phosphorylation
Origin of mitochondria
bacteria
- single event where primitive eukaryote engulfed a bacterial cell, giving rise to possibility for complexity in organisms (e.g. multicellularity)
Functions of Mitochondria
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;
Features of Mitochondrial DNA (mtDNA)
- 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
How many genes are in mtDNA?
37 genes
- 13 encode oxidative phosphorylation protein subunits (OXPHOS proteins)
- 22 encode tRNAs
- 2 encode rRNAs
OXPHOS proteins
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
Function of respiratory chain complexes
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
What is a D-loop in mtDNA?
non-coding region where replication and transcription are initiated and contains regulatory sequences for this
Where is mitochondria inherited from?
thought to be strictly maternally inherited and there is no recombination
What does the mitochondrial genome encode?
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
What does the non-coding region (D-loop) of mtDNA contain?
regulatory sequences important for mitochondrial replication and transcription
Where does mtDNA replication and transcription start from?
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)
What is the mtDNA packaged into?
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
Exceptions in mtDNA to the “universal” genetic code
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)
mtDNA haplogroups
no recombination in mtDNA and maternally inherited so therefore mutations acquired over time subdivide the human population into discrete haplotypes
Requirements of mtDNA to produce OXPHOS proteins
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
mtDNA replication machinery
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
mtDNA Polymerase
Polymerase Gamma:
-heterotrimeric protein
What are the subunits of polymerase gamma?
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
mtDNA TWINKLE
TWINKLE is mtDNA helicase with 6 different subunits (hexamer)
- unwinds double stranded mtDNA template to allow replication by polymerase gamma
mtDNA Single Stranded Binding Protein (mtSSBP)
Binds single stranded DNA to:
- protect against nucleases
- prevents secondary structure formation
- enhances mtDNA synthesis by stimulating TWINKLE helicase activity
Which model represents mtDNA replication?
Strand displacement model
Strand displacement model of mtDNA replication
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)
