The mitochondrial genome Flashcards
What are mitochondrial functions?
- Haem Synthesis
- Generates ATP
- Apoptotic cell death
- Produces ROS
- Important in inflammation and innate immunity, surface of antibodies contain antibodies called the mitochondrial antiviral signalling complex/ MAVs
What is the structure of the mitochondrial genome?
- Double stranded circular molecule
- Consists of heavy and light strand
-
Multicopy genome (10-100,000 copies per cell)
- 37 genes
- 13 oxphos protein subunits
- 22 transfer RNAs
- 2 ribosomal RNAs (required for translation of 13 encoding protein subunits)
- 37 genes
- No INTRONS
- D-loop is non-coding region where replication and transcription are initiated
- Maternally inherited, no recombination
What does the mitochondrial genome consist of and not have?
Consists of heavy and light strands and has no introns
How many genes does the mitochondrial genome have?
Has 37 genes
What are the genes present in the mitochondrial genome and the amount present?
-13 Oxidative phosphorylation protein subunits -22 tRNAs -2 rRNAs
What is the D loop in the mitochondrial genome?
Comment on processes and where they occur in mitochondrial DNA
The D loop is a non-coding region where replication and transcription are initiated
- mtDNA replication starts in Origin of heavy strand (OH)
- Transcription starts at Heavy strand promoter (HSP) and light strand promoter (LSP)
How is the mitochondrial genome inherited?
Maternally inherited, no recombination
What does the mitochondrial genome encode?
Encodes proteins of oxidative phosphorylation
- There are 13 OXPHOS proteins
- The first four are respiratory chain complexes (CI-CIV) and CV is ATP synthase
What does the non-coding region contain?
Contains regulatory sequences for replication and transcription
What is mtDNA packaged into?
Packaged into nucleoids
- There are 1 or 2 copies of mtDNA per nucleoid
-
Transcription factor A (TFAM) acts as a histone protein
- Prevents it from being damaged and replicated
-
Transcription factor A (TFAM) acts as a histone protein
Explain how mtDNA gives rise to haplotypes
Because mtDNA is maternally inherited it will give rise to haplotypes
Haplotypes = Group of alleles inherited together from a single parent
-
mtDNA does not recombine, and mutations acquired over time subdivide the human population into discrete haplogroups
- This is used to track migration of human populations
- e.g mtDNA originally had main lineages L0 to L3, these gave rise to MNN which were then taken to different parts of the world giving rise to sub haplogroups
What does transcription factor A(TFAM) act as when mtDNA is packaged?
Acts as a histone protein
Genetic code in vertebrate mitochondria DNA vs nuclear DNA
-AUA and AUG code for methionine in mitochondria; whereas in nuclear DNA, AUA codes for isoleucine -UGA codes for tryptophan in mitochondria; whereas in nuclear DNA, it’s a stop codon -AGA and AGG are stop codons in mitochondria; whereas in nuclear DNA, it’s arginine
What is the endosymbiotic theory of the origin of the mitochondria?
- Primitive eukaryotic cell ingested a bacterium and the bacterium survived, the two organisms benefitted through a symbiotic relationship.
- The bacterium evolved to be what we now call mitochondria
What does the mitochondria require both of?
Requires both nuclear and mtDNA encoded proteins
How many proteins does oxidative phosphorylation require?
Oxidative phosphorylation requires >100 proteins but mtDNA only encodes 13 of those proteins
What must happen in order to make the 13 proteins for oxidative phosphorylation?
In order to make the 13 proteins for oxidative phosphorylation, protein mtDNA must be:
- Replicated
- Transcribed
- Translated
What are all the proteins involved in replication, transcription and translation of mtDNA encoded by?
All proteins involved in replication, transcription and translation of mtDNA are encoded by nuclear genes and imported into mitochondria
What are all the other proteins involved in oxidative phosphorylation made by?
All other proteins involved in oxidative phosphorylation are made by nuclear genes
What do you need to replicated mtDNA?
- 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.
Describe the mtDNA polymerase
What is the function of POLgA and POLgB?
Called
- Polymerase gamma (POLG)
-
Heterotrimer protein (requires two nuclear genes)
- 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.
What does POLgammaB enhance?
Enhances interactions with DNA template and increases activity and processability of POLgammaA
What is the structure of mtDNA helicase(TWINKLE)?
TWINKLE: -Six TWINKLE subunits
Unwinds the double stranded mtDNA template and allows replication by POLG
What does the mitochondrial single stranded binding protein (mtSSBP) bind to and what does it do?
Binds to single stranded DNA
- -Protects against nucleases
- -Prevents secondary structure formation
- -Enhances mtDNA synthesis by stimulating TWINKLE helicase activity
Steps involved in the replication of mtDNA
- Replication of heavy strand
- Replication of light strand
- Replication of both strands complete and segregation of daughter molecules occurs
Origin of the light chain allows the replication to occur in the opposite direction
Replication of heavy strand
- -Parental heavy strand displaced and coated with mtSSBP
- -TWINKLE helicase unwinds mtDNA
- -Mitochondrial polymerase (POLRMT) synthesizes RNA primer using light strand as template
- -POLgamma uses RNA primer to replicate DNA at OH
Replication of light strand
- -Heavy strand replication passes OL
- -Stem loop structure is formed preventing mtSSBP binding
- -Mitochondrial polymerase (POLRMT) synthesizes RNA primer using heavy strand as a template
- -POLgamma uses RNA primer to replicate light strand DNA at OL
Completion of both strands
-Synthesis proceeds until both strands are fully replicated -After replication, daughter molecules are segregated by topoisomerase 3A
Why are mtDNA strands named heavy and light strands?
Heavy chain has more purines which are larger than pyrimidines
Mitochondrial diseases
- Rare monogenic diseases
- Affect highly metabolic organs
- Can affect one or several organ systems
- Genetically heterogeneous
What causes mitochondrial disease?
Mutation in >300 nuclear genes cause mitochondrial disease, not just mtDNA
What determines mitochondrial disease manifestation?
Heteroplasmy levels determine mitochondrial disease manifestation
How is mtDNA inherited and mutation load?
- mtDNA is maternally inherited
- inheritance of mutation load is random (one individual may have a low mutation load however offspring may have a higher load)
Since we know there are nuclear genes which encode mitochondrial proteins, you will expect to see autosomal recessive patterns of inheritance of mitochondrial diseases, dominant X-linked, de novo patterns
What way can be used to identify mtDNA mutations
mtDNA mutations can be identified by next generation sequencing
