1. Presented
by
Upasana Ganguly

2. Introduction
 Mitochondria are eukaryotic organelles involved in
many metabolic pathways, but their principal function
is the generation of most of the cellular ATP through
the oxidative phosphorylation system (OXPHOS).
 Mitochondria are unique organelles since they require
the contribution of two physically separated genomes.
 The pioneering work of George Palade and coworkers
developed a protocol for the isolation of mitochondria
based on differential centrifugation.

3. Structure of Mitochondria
 Mitochondria range from 0.5 to 10 micrometers in
diameter.
 Mitochondria are surrounded by a double membrane
system – the outer and the inner mitochondrial
membrane.
 The outer membrane contains proteins called porins.
 The inner membrane has a high proportion of proteins
and “double” phospholipid cardiolipin.
 The inner membrane forms numerous folds called cristae
which extend into the matrix of the mitochondria.
 The matrix contains the mitochondrial genetic system as
well as the enzymes responsible for the oxidative
metabolism.

4. Fig.1 (A)cross section, as seen in the electron microscope. (B) A
drawing of a mitochondrion with part of it cut away to show the
three-dimensional structure. (C) A schematic eukaryotic cell,
with the interior space of a mitochondrion.

5. The Genetic System of Mitochondria
 Mitochondria contain their own genetic system which is
separate and distinct from the nuclear genome of the cell.
 The existence of a separate mitochondrial genome is
explained by the widely accepted endosymbiotic theory
according to which the mitochondrion developed from an
α-proteobacterium.
 During the course of time ancient bacterial genes may have
been transferred from the mitochondrial to the nuclear
genome.
 There are open questions regarding why mitochondria have
retained their genetic material and why elaborate
enzymatic machineries are required to replicate and
express a separate genome containing only a few genes.

6. Basic features of Mitochondrial genome
 Mitochondrial genomes are circular, double stranded
DNA molecules which is about 16,600 bp in humans
and are present in multiple copies per organelle.
 The largest sequenced mitochondrial genome is of the
plant Arabidopsis (367 kb )and the smallest is of the
protist Plasmodium falciparum (6kb).
 The largest number of mitochondrial genes has been
found in the mtDNA of the protozoan Reclinomonas
americana.
 mt DNA encode 13 of the ~ 90 different proteins
present in the respiratory chain of mammalian
mitochondria, 2 rRNAs and 22tRNAs.

7. Peculiarities of the Mitochondrial
genome
 Cells are polyploid with respect to mitochondria.
 Mitochondrial genome is maternally inherited.
 mtDNA is continuously turned over and replicated
throughout the entire cell cycle with no distinct phase
specificity.
 The evolution rate of mtDNA is much faster than that
of the nuclear genome.
 Mitochondrial genes are translated using a genetic
code with some differences from the universal genetic
code.

8. Structure of mt DNA
 The individual strands of the mtDNA molecules
are denoted heavy (H) strand and light (L) strand
because of their different buoyant densities in CsCl
gradient.
 mtDNA is a supercoiled structure and is poorly
associated with proteins.
 The compact mammalian mtDNA lacks introns
and the only longer non-coding region (D-loop)
contains the control elements of transcription and
replication.

9. The Organization of Human
mitochondrial genome
Fig.2 The organization of human mitochondrial genome : The genome
contains 13 protein coding genes,2 rRNA genes and 22 tRNA genes.

10. mtDNA mutations
 A pathogenic mutation may be present in all copies of
mtDNA (homoplasmy) or only in a fraction of all copies
(heteroplasmy).
 Heteroplasmic mtDNA mutations seggregate during cell
division.
 A minimum threshold level of mtDNA mutation must be
present in a cell to cause respiratory chain deficiency
ranging from 50-60% for mtDNA deletions and >90% for
some tRNA mutations.
 Transmission of heteroplasmic mtDNA mutations from
mother to child is rare whereas transmission of point
mutations is common among human pedigrees.
 Some disorders known to be associated with mtDNA
mutations : MELAS, MERRF, Kearns-Sayre-CPEO ,Leber’s
hereditary optic neuropathy (LHON), Aminoglycoside-
associated deafness, Diabetes with deafness.

11. mt DNA transcription and RNA
processing
1.Structural features of mtRNAs
2.Mitochondrial RNA Polymerase
3.Mitochondrial Transcription factors
4.Mechanism of Transcription
5.RNA processing and maturation

12. 1.Structural features of mtRNAs
 rRNAs are smaller than cytoplasmic or bacterial
rRNAs, methylated and contain a short 3´ poly(A) tail
of 1–10 residues.
 Mitochondrial tRNAs are also smaller (59–75nt) than
their cytoplasmic counterparts and have some
structural differences.
 tRNAs lack the so-called constant nucleotide positions
and the size of the ‘DHU’ loop is very variable.
 mRNAs start directly at the initiation codon or have an
extremely short untranslated 5´end and contain a
polyA tail of 55 residues immediately after the stop
codon.

13. 2. Mitochondrial RNA Polymerase
 Single subunit enzyme.
 Human POLRMT gene encodes a protein of 1230 amino
acid residues including a 41-residue N-terminal targeting
peptide.
 C-terminal part of the protein contains a series of
conserved motifs.
 Unique N-terminal extension with unknown function.
 N-terminal extension contains a putative pentatricopeptide
repeat (PPR) which is 35 amino acids long.
 POLRMT cannot interact with promoter DNA and initiate
transcription on its own and requires the assistance of
mitochondrial Transcription factors (mtTFs).

14. 3.Mitochondrial Transcription factors
 mtTFB1 and mtTFB2 form heterodimeric complex with
POLRMT.
 They display sequence similarity to a family of rRNA
methyl transferases.
 mtTFA directly regulate the activity of both
mtTFB1/POLRMT and mtTFB2/POLRMT-dependent
mtDNA transcription in vitro.
 mtTFA contains two HMG box domains separated by a
linker region and followed by a C-terminal tail.
 mtTFA can bind, unwind and bend DNA without sequence
specificity.

15. 4. Mechanism of transcription
 Each strand contains a promoter for transcriptional
initiation called HSP and LSP.
 HSP transcription is initiated from two specific sites
:H1 and H2.
 Binding of TFAM introduces specific structural
alterations in mtDNA causing unwinding of promoter
and transcription initiation.
 POLRMT contains a “specificity loop” which is
inserted into the DNA major groove.
 POLRMT-mtTFB2 heterodimer protects +10 to -4
region of light strand promoter (LSP).
 Transcription termination at H1 site occurs by binding
of mTERF protein to a 28-bp region at the 3´end of
tRNALeu.

16. Fig.3 Schematic representation of the mammalian D-loop and
transcription termination regions, showing the main elements and
factors involved in transcription and in replication initiation.

17. 5.RNA processing and maturation
 Transcription from the mitochondrial promoters
produce polycistronic precursor RNAs which must be
processed to produce individual mRNA, rRNA and
tRNA molecules.
 According to this model of ‘tRNA punctuation’, tRNA
sequences located between each rRNA and mRNA
would act as the signals for the processing.
 This processing requires at least four enzymatic
activities:
i) tRNA 5´ and 3´ end endonucleolytic cleavages
ii)a polyadenylation activity for rRNAs and mRNAs
iii) the addition of the CCA to the tRNA 3´ end.

18. mtDNA replication
1. mtDNA Polymerase γ
2. mt TWINKLE Helicase
3. mt SSB
4. Mechanism of replication

19. 1.mtDNA Polymeraseγ
 First identified as RNA dependent DNA Polymerase in
Human HeLa cells.
 Distinguished from other cellular DNA Polymerases by a
number of chemical criteria including high activity using
synthetic RNA templates in vitro and resistance to
aphidicolin.
 Belongs to family A group of DNA Polymerases.
 Catalytic subunit POLγA has a molecular mass of 140KD
and has polymerase,3´-5´exonuclease and 5´deoxyribose
phosphate lyase activities.
 POLγA is associated with a smaller subunit of 55KD called
POlγB which shares a high level of structural similarity to
class IIa family of tRNA synthetases.
 POLγB subunit substantially increases both the catalytic
activity and processivity of POLγA.

20.  mt TWINKLE Helicase first identified by positional
cloning.
 It has been found mutated in some cases of
Progressive External Opthalmoplegia (PEO),a human
disorder associated with multiple mtDNA deletions.
 The protein catalyses the ATP-dependent unwinding
of a DNA duplex with a distinct polarity (5´ to 3´).
 Preferred substrate resembles a DNA replication fork.
2. mt TWINKLE Helicase

21. 3. Mitochondrial Single-Stranded DNA binding
protein
 mt SSB has a molecular weight of 13-16KD.
 Displays sequence similarity to E.coli SSB.
 Forms tetramer.
 Binds co-operatively to DNA with a binding site
size of 50-70 nucleotides per tetramer.
 Has a stimulatory effect on the rate of unwinding
by TWINKLE.

22. 4. Mechanism of replication
 Exact model of replication is still debated.
 POLRMT provides the primer needed for DNA
replication by stimulating transcription from LSP.
 To origins of replication : OH and OL
 When leading strand synthesis from OH proceeds to
2/3rd of the genome it exposes the OL and lagging
strand synthesis initiates.
 New mtDNA molecules are ligated to form closed
circles.
 Superhelical turns are induced.

23. Strand-displacement model of mtDNA replication shown
in progress, just after the beginning of synthesis of a new
light strand from OL
Broughton R E , Reneau P C Mol Biol Evol 2006;23:1516-1524
Fig4.Strand-displacement model of
mtDNA replication shown in
progress, just after the beginning of
synthesis of a new light strand
from OL. Replication begins
at OH and the original heavy strand
is displaced and becomes single
stranded as the polymerase complex
passes, proceeding clockwise in this
orientation. The heavy strand is
then made double stranded as
another polymerase complex
proceeds back in the opposite
direction, moving counterclockwise
from OL. The positions of the ND2,
COI, and Cytb genes are labeled for
reference.

24. Conclusion
 Mitochondrial biology has been studied
extensively for decades using traditional
biochemical and molecular approaches.
 Despite great progress in the field, many aspects of
mitochondrial biogenesis remain to be fully
elucidated.
 There is no generally accepted model for mtDNA
replication and new experimental approaches
should be used to conclusively resolve this issue.

25. THANK YOU