MATH 560 Lecture 13: Mitochondria notes 13 March
29 Jun 2018
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Mitochondria-PART I
• Small organelles
• Has a lot of transporters and pumps
• Mitochondrial cristae (finger like extensions) present in the inner membrane of the
mitochondria-increases the surface area of the membrane
• They are called the powerhouse of the cells- ATP synthesis (except red cells do not have
mitochondria), ROS generation (consequence of ATP synthesis, they are signaling molecules,
ROS in excess can be toxic)
• Regulators of apoptosis- when a cell gets a signal to kill itself, molecules like Cytc is released
from mitochondria
• Fatty acid metabolism- except VLCFA (VLFCA are metabolized in peroxisomes)
• Fe-S centre biosynthesis- redox enzyme centres, a whole new biosynthetic apparatus is present
for this
• Urea cycle
• Heme biosynthesis
Most mitochondria diseases occur due to impaired ATP synthesis
They synthesize ATP in the OXPHOS machinery-5 complexes involves-electrons in the form of NADH get
transferred between the complexes- free energy is released to generate protons- protons are released
in a cautious way from the last complex and is coupled with phosphorylation of ADP to form ATP
You need two genomes to put this process together- four of the complexes have structural units
encoded by the mitochondrial genome, rest of the components are encoded by the nuclear DNA
• Component 2 has all its 4 subunits encoded by the nuclear DNA
• All the other components have mitochondrial encoded structural units.
• Mammalian mtDNA-Small double stranded molecule, 16.5 kilobases, codes 37 genes, codes 13
proteins (All essential), two ribosomal RNA’S, 22 tRNA, no introns, most of them do not have a
stop codon (maybe because of some evolutionary pressure) and need to be polyadenylated to
stop expression sometimes. There are two strands- heavy and light strands (can be separated by
Cs-Cl density gradient due to GC content bias)- termed as ‘heavy’ and ‘light’ for historical
reasons
• Proteins coded by mitochondrial DNA are highly hydrophobic and have transmembrane
domains – these proteins need to be made in the tissues they are needed in.
• There is no individual promoter elements. Transcription apparatus is very simple. Genetic code
is different form the universal code.
• Because mitochondrial DNA has a stop codon that is different from nuclear DNA, it cannot
employ the cell’s ribosome for translation.
(Mitochondria is said to originate from bacteria- a lot of genes in the bacteria were lost over time)
There is a special type of polymerase-polymerase gamma-only targeted for mitochondrial DNA
replication. Mutation rate is 10 times greater than nuclear genes. This is why every person has a
mitochondrial genomic variation of at least 50 bp. That is why mitochondrial genes are studied to
understand evolution.
Mitochondria is organized into nucleoids (like the bacterial genome). Protein DNA compelxes associated
with the mitochondrial membrane e.g tfam (most important for packing the genome-Tfam is most
important factors in packing, can pack on its own.) Genome is organized like beads on a strand.
Role of Tfam for mitochondrial DNA function (Important)- the mitochondrial transcription factor TFAM,
an abundant and highly conserved High Mobility Group box protein, binds DNA cooperatively with
nanomolar affinity as a homodimer and that it is capable of coordinating and fully compacting several
DNA molecules together to form spheroid structures. We use noncontact atomic force microscopy,
which achieves near cryo-electron microscope resolution, to reveal the structural details of protein–DNA
compaction intermediates. The formation of these complexes involves the bending of the DNA
backbone, and DNA loop formation, followed by the filling in of proximal available DNA sites until the
DNA is compacted. These results indicate that TFAM alone is sufficient to organize mitochondrial
chromatin and provide a mechanism for nucleoid formation.
Figure 1 Purified Tfam was subjected to nanoscopic atomic force microscopy and Electrophoretic mobility shift assay (EMSA)
Mitochondrial Transcription
• Initiation (doi: 10.1074/jbc.C110.128918)-Human mitochondrial genome contains two
promoters located in the opposing DNA strands. (H and L)
• The HSP1 promoter is responsible for synthesis of most mitochondrial genes and is activated by
POLRMT-TFAM-TFB2M complex.
• During replication, when DNA region near oriL becomes single-stranded and forms stem-loop
structure, transcription by POLRMT generates short RNA primers. This initiation event is TFAM-
and TFB2M-independent.
• Transcription from the LSP promoter generates primers for replication at oriH as well as the rest
of the tRNAs and mRNA.
• This initiation event, like HSP1, requires cooperative action of POLRMT, TFAM, and TFB2M for
efficient transcription and replication.
• The H-strand is transcribed by two overlapping units. One of them starts at the initiation site H1,
located 19 nt upstream of the tRNA
• Phe gene and ends at the 16S rRNA3‚-end. This transcription unit operates much more frequently
than the second one and is responsible for the synthesis of the two ribosomal RNAs, tRNAPhe and
tRNA Val
• The activity of this unit is linked to a transcription termination event taking place immediately
downstream from 16S rRNA, inside the gene for tRNALeu
• The second transcription unit, operating with a frequency about 20 times lower, starts at
• the initiation site H2, close to the 12S rRNA 5‚-end and originates a polycistronic molecule
covering almost the whole H-strand. The mRNAs for the 12 H-strand encoded
• polypeptides and 12 tRNAs derive from the processing of this polycistron. This transcription model
explains how a differential regulation of rRNA versus mRNA transcription can be operated
through the initiation of H-strand
• transcription at the two alternative sites (Montoya et al,1983)
• The L-strand gives rise to a single polycistron starting at the 5‚-end of 7S RNA, about 150 bp away
from the H1initiation point, from which the eight tRNAs and the ND6 mRNA are derived.
• The two-major mammalian mitochondrial promoters containing the H1and L initiation points,
called HSP1andLSP, respectively, are functionally independent and have a bipartite structure.
• First, they contain a promoter element with a consensus sequence motif of 15 bp which surrounds
the initiation points and is essential for transcription.
• RNA processing and maturation:
(https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/eph8802514)
• RNA sequences located between each rRNA and mRNA would act as the signals for the processing
enzymes after acquiring the cloverleaf structure on the nascent RNA chains.
• This processing requires at least four enzymatic activities:first, the tRNA 5‚ and 3‚ end
endonucleolytic cleavages;
• second, a polyadenylation activity for rRNAs and mRNAs
• Finally, the addition of the CCA to the tRNA 3‚ end.
• The 5‚endonucleolytic cleavage occurs first and is performed by a mitochondrial RNase P as had
been previously suggested
• This kind of enzyme also exists in the nucleus and is usually a ribonucleoprotein with an essential
RNA component
• Polyadenylation is performed by a mitochondrial poly(A) polymerase and plays an important
role in stabilizing the RNAs and in many cases it even contributes to the generation of the stop
codons of some mRNAs
• There is evidence that the polyadenylation of mRNAs occurs during or immediately after
cleavage.
• RNaseP cuts at 5’ end of tRNA. RnaseZ cuts at 3’ end of tRNA.
• Human mtRNA polymerase (mtRNApol or POLRMT), a 120 kDa protein, which has recently been
cloned, is homologous with phage polymerases and yeast mtRNA polymerases
• In the absence of transcription factors, the mtRNApol is unable to recognize the mitochondrial
promoters and shows little and only non-specific activity
• tfam like other HMG family proteins, is able to wrap, bend and unwind DNA in vitro with a low
degree of sequence specificity
