SINH HỌC PHÂN TỬ

Molecular biology of mitochondria
Mitochondria are the main site of ATP synthesis in eukaryote cells and as such are vital for the health and survival of the cell

They are also one of the sites at which apoptosis is mediated
These lectures will explore the molecular genetics of mitochondria, how they are made, the structure of their genome, how they evolved , and how mitochondrial gene expression is controlled.
Molecular Biology 325 2007
Mitochondrial molecular genetics 1

• focus on mitochondria: brief overview of their function and structure
• mtDNA structure and replication:
- animals
- yeast
- plants
• inheritance of mitochondria
- petite mutants of yeast
• biogenesis of mitochondria by fission
MITOCHONDRIA


• essential for cell life

- ATP synthesis

- many metabolic intermediates
• essential for cell death

- unprogrammed death: necrosis
( eg, due to loss of energy status)

- programmed cell death
(apoptosis - controlled cell destruction)
• Two membranes
• Inner membrane invaginated
• Numbers of mitochondria per cell
vary but usually 100s/cell
Matrix contains the TCA cycle (and other) soluble enzymes

Inner membrane contains metabolite transporters and the electron transport chain
Mitochondrial structure
Overview of aerobic respiration
NADH
+ CO2
One pyruvate molecule is completely oxidised to CO2
4-Carbons
3-Carbons
CO2
6-Carbons
NADH + CO2
NADH
FADH
Outline of Tricarboxylic Acid Cycle
The NADH and FADH produced are oxidised by the respiratory electron transport chain
Four large, multi-subunit protein complexes

- complex I is a NADH-ubiquinone reductase
- complex II is succinate dehydrogenase (part of the TCA cycle)
- complex III is the ubiquinone -cytochrome c reductase
- complex IV is cytochrome oxidase
The respiratory electron transport chain
Mitochondria have their own DNA and Ribosomes

Mitochondria have some of their own DNA, ribosomes, and can make many of their own proteins. The DNA is circular and lies in the matrix in structures called "nucleoids".  Each nucleoid may contain 4-5 copies of the mitochondrial DNA (mtDNA).
mitochondrial
DNA
The ribosomes can actually be visualized in some mitochondria. In these figures, they are seen in the matrix as small dark bodies. DNA can also be visualized in mitochondria. The DNA is circular and resembles that of a bacterium in its basic structure.
Mitochondria also have their own ribosomes and tRNA:
• 22 tRNAs
• rRNAs (16S and 12S)
To visualize the structure of mitochondrial DNA, we have to extract the DNA and float it on a water surface. Then, it can be picked up by a plastic coated grid, and examined in the electron microscope. Mitochondrial circular DNA is shown in the figure.
Mitochondrial Inheritance


Yeast has been used extensively to study mitochondrial inheritance.

There is a Yeast strain, called "Petite" that have structurally abnormal mitochondria that are incapable of oxidative phosphorylation.  These mitochondria have lost some or all of their DNA.

Genetic crosses between petite and wt strains showed that inheritance of this trait did not segregate with any of the nuclear chromosomes.
Mitochondrial inheritance from yeast is biparental, and both parent cells contribute to the daughter cells when the haploid cells fuse.  After meiosis and mitosis, there is random distribution of mitochondria to daughter cells.  If the fusion is with yeast that are petite and yeast that are not, a certain percentage of the daughter cells will be "petite".  
Mitochondrial Inheritance
Mitochondrial Inheritance in Yeast
This led to the suggestion that some genetic element existed in the cytoplasm and was inherited in a different manner from nuclear genes. This is called “non-Mendelian inheritance” or “cytoplasmic inheritance”.
Mitochondrial Inheritance
In yeast and animals, this indicated inheritance of mitochondrial genes: in plants it also includes inheritance of chloroplast genes
Mitochondrial replication
Mitochondria replicate much like bacterial cells. When they get too large, they undergo fission. This involves a furrowing of the inner and then the outer membrane as if someone was pinching the mitochondrion. Then the two daughter mitochondria split. Of course, the mitochondria must first replicate their DNA. An electron micrograph depicting the furrowing process is shown in these figures.
Mitochondrial replication
Sometimes new mitochondria are synthesized in centres that are rich in proteins and polyribosomes needed for their synthesis. The electron micrograph in the following figure shows such a centre. It appears that the cluster of mitochondria are sitting in a matrix of proteins and other materials needed for their production.
Certain mitochondrial proteins are needed before the mitochondria can divide.

This has been shown in a study by Sorgo and Yaffe, J Cell Bio. 126: 1361-1373, 1994. They showed the result of the removal of an outer membrane protein from mitochondria called MDM10. This figure shows the results. The mitochondria are able to take in components and produce membranes and matrix enzymes. However, fission is not allowed and the result is a giant mitochondrion.
giant
mitochondrion
Human mtDNA

• small, double stranded
circular chromosome
• 16,569 bp in total
• no non-coding DNA
• no introns
• polycistronic replication
which is initiated from
the D (displacement)- loop
region
• followed by splicing of
transcript to form
messages.
Organisation of the mitochondrial chromosome
human mtDNA
yeast mtDNA
Yeast
mitochondrial
chromosome
Human DNA

• 16,569 bp;
• no non-coding DNA
• no introns
• polycistronic replication followed by splicing to form messages.

Yeast mtDNA
• 68-75 kb, similar in structure to bacterial genome
• contains introns and non-regions between genes.
• Same proteins made as in animals
• genes transcribed separately
Despite having their own genome, most mitochondrial proteins are encoded in the nucleus, made in the cytosol and imported into the mitochondria
In all organisms, only a few of the proteins of the mitochondrion are encoded by mtDNA, but the precise number varies between organisms

• Subunits 1, 2, and 3 of cytochrome oxidase
• Subunits 6, 8, 9 of the Fo ATPase
• Apocytochrome b subunit of complexIII
• Seven NADH-CoQ reductase subunits (except in yeast)

The nucleus encodes the remaining proteins which are made in the cytosol and imported into the mitochondrion.

Most of the lipid is imported.
Synthesis of mitochondrial proteins
Plant mtDNA
• chromosome size is much bigger but varies dramatically between species (200-2000 kb)
• arranged as different size circles, sometimes with plasmids.
• The plant mtDNA contains chloroplast sequences, indicating exchange of genetic information between organelles in plants.
• Much of the plant mtDNA is non-coding, but coding regions are larger than animals and fungi.
• Number of proteins synthesised not known definitely but more than in animals and yeast (probably about 50)
Plant mitochondria have specialised functions
• in leaves they participate in photorespiration
• sites of vitamin synthesis (vit C, folic acid, biotin)
maize mitochondrial genome
In plants, respiration and photosynthesis operate simultaneously in the light
NIGHT
DAY
Chloroplasts are the site of photosynthesis and belong to the plastid family of organelles - they develop from proplastids in the light
proplastid
Rice mitochondrial and chloroplast genomes

Plant mitochondria contain chloroplast genes - suggesting that genetic transfer occurs between the two organelles
Mitochondrial DNA of animals and fungi uses a different genetic code
than the “universal” code
RNA processing in mitochondria
Plant mitochondria “edit” their RNA transcripts. This was first noticed when comparing cDNA sequences with genomic DNA sequences.

The most common change is to replace C with U, although in some instances other changes can occur. Matrix enzymes are thought to be responsible for this, but the reason for the editing is not known.

Most of the DNA in plant mitochondria is non-coding, only some of which is transcribed. RNA editing occurs even in non-coding regions such as introns.
Evolution of mitochondria
Mitochondria are generally thought to have evolved endosymbiotically when an anaerobic prokaryotic cell engulfed an aerobic bacterium and formed a stable symbiosis. Loss of most of the aerobe’s genome to the nucleus of the host allowed the latter to control the former.
This is supported by gene sequence analysis which shows remarkable homology between bacteria and mitochondrial genes.