An Overview of Mitochondrial Biology

Presented by

Upasana Ganguly

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.

Structure of MitochondriaMitochondria 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.

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.

The Genetic System of MitochondriaMitochondria 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.

Basic features of Mitochondrial genomeMitochondrial 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.

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.

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.

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.

mtDNA mutationsA 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.

mt DNA transcription and RNA processing

1.Structural features of mtRNAs2.Mitochondrial RNA Polymerase3.Mitochondrial Transcription factors4.Mechanism of Transcription5.RNA processing and maturation

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.

2. Mitochondrial RNA PolymeraseSingle 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).

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.

4. Mechanism of transcriptionEach 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.

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.

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.

mtDNA replication1. mtDNA Polymerase γ 2. mt TWINKLE Helicase 3. mt SSB4. Mechanism of replication

1.mtDNA PolymeraseγFirst 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.

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

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.

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 OLWhen 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.

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.

ConclusionMitochondrial 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.

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