Mitochondrial Disease Flashcards
What are mitochondria?
They are the powerhouse of the cell, through OXPHOS and the TCA cycle provide the energy in the cellular currency of ATP
Describe mitochondrial DNA
MtDNA is circular and is composed of the heavy and light chain. It encodes 13 core proteins, 22 tRNAs and 2 rRNAs (12S and 16S) and others, ecompassing 37 genes.
The encoded “core” proteins compose critical subunits of Complexes I, III-V.
There are between 2-10 copies per cell and the mtDNA is composed of 16,569 nt.
There are no introns therefore no 3’ or 5’ UTRs, thus upon transcription a polycistronic strand will be produced, cleavage occurs either side of tRNAs.
Describe mtDNA transcription
Due to a lack of introns they are transcribed polycistronically, with transcription being initiated at the D loop.
The initiation of transcription required the binding of TFAM upstream of the HSP.
Transcription requires the mtRNA polymerases.
Transcription of the HS usually is initiated at the ITH1 and is terminated at the tetradecamer in the tRNAleu gene, this is called the mtTERM site.
IH2 transcription is less frequent and doesnt require TFAM - however it still generates a transcript (polycis)
Where are the promoters on the heavy and light strand?
Heavy - ITH1 (HSP), ITH2 (tRNAphe)
Light - LSP
Describe the processing of the mtRNA polycistronic transcript.
tRNAs are typically interspersed between the protein and rRNA transcripts and their secondary structure acts as a recognition site for endonucleases.
What cleaves the 5’ end in polycistronic transcript processing and the 3’ end?
5’ - mRNase P
3’ - tRNA endonuclease
What adds the CCA discriminator sequence to the tRNAs?
ATP(CTP):tRNA nucleotidyl transferase
Describe mtDNA replication
It is independent of nuclear replication, however the regulatory mechanisms governing mtDNA replication are currently unclear.
Nuclear factors are required for mtDNA replcation, namely PolG.
- Origin of H strand replication (oh) is were replication begins, a primer is added by the LSP
- nDNA derived PolG extends the sequence passed the free 3’OH of the primer typically at the Conserved Sequence Block 1, the unwinding of the mtDNA downstream of polG is aided by DNA TWINKLE (helicase)
- Single Stranded Binding proteins are sequentially added to the strands to ensure separation and uninterupted replication
- The termination site is at the termination associated sequence this allows for secondary structure formation of the D loop
- Before replication termination primers will be cleaved from the sequence by RNase MRP (mitochondrial RNA processing) and endonuclease G
Why are mtDNA suscupetible to mutation?
They are held in nucleoids, meaning they are not protected by histones.
They are additionally in close proximity to ROS generated by the MRC, this could lead to deamination of adenine, the infliction of double strand breaks, addition of hydroxyl radicals tp guanine generating 8-oh-guanine
What mechanisms are in place to control/prevent DNA damage
Previously there were thought to be no “maintenance” systems in place, it is now known that mtDNA can repair itself through BER - this can repair 8-oh-guanine. However it does lack NER, and mismatch repair.
Additionally PolG has not only polymerase capabilities but a 3’ –> 5’ exonuclease domain.
What is the mtDNA helicase
TWINKLE TWINKLE LITTLE STAR
Where is replication terminated
Often at the termination associated sequence (TAS) here forms the D loop secondary structure
What cleaves the primers
Endonuclease G and RNase MRP (mito RNA processing)
What is upstream of the DNA primer?
Typically downstream of the Conserved Sequence Block 1 (CSB1)
What can inhibit mtDNA translation
Chloramphenicol - this demonstrates the similarities with prokaryotic translation
