Lecture 6: Mitochondrial Genetics in Health Flashcards
What happened 3.6 billion years ago…? …6
- The first signs of life began about 3.6 billion years ago, in the OCEAN
- The iron-rich rocks formed before the presence of atmospheric oxygen, and life itself. Found upon these rocks are 3.45 billion-year-old fossil STROMATOLITES, COLONIES OF MICROBIAL CYANOBACTERIA.
- The first signs of life began about 3.6 billion years ago, in the ocean.
- The water in the ocean protected organisms living there from the dangerous rays of the sun.
- These x-rays and gamma-rays are part of the electromagnetic spectrum, which damage living things.
- Later, Earth developed an atmosphere.
LIFE ON EARTH …2 BILLION YEARS AGO…6**
- Primitive Heterotrophic Eukaryote
and
and Autotrophic Alpha Proteobacterium Prokaryote
exits
- Eukaryote EATS but DOES NOT DIGEST Prokaryote
- Eukaryote keeps BACTERIUM as a SYMBIONT
- Symbiont loses many genes and becomes MITOCHONDRION
- LIFE EVOLVED TO AS WHAT WE KNOW NOW
Mitochondria are essential for life… EXPLAIN …3
1 * Produce >95% of cellular energy
by oOXIDATIVE PHOSPHORYLATION
2 * Mitochondria produce 64 KILOGRAMS of ATP EVERY DAY
3 * SUPPORT MOST of the ACTIVITIES OF OUR CELLS
Mitochondrial how shoes it look like? how much? what does it do?. EXPLAIN …5
1 * MITOCHONDRIA can have DIFFERENT SHAPES IN DIVERSE CELL TYPES AND ORGANS
- TheNUMBER OF MITOCHONDRIA DIFFERS BETWEEN ORGANS AND CELL TYPES …. most of the time depending on energy requirements
3 * MITOCHONDRIA FORM RETICULAR NETWORKS THROUGH RETICULAR NETWORKS THROUGH WHICH THEY COMMUNICATE WITH EACH OTHER.
4 * MITOCHONDRIA ASSOCIATE WITH OTHER ORGANELLES SUCH AS THE ER
5 *MITOCHONDRIA UNDERGO FUSION AND FISSION
Mitochondria are the powerhouse of the cell…
Features of the Mitochondria…3
1 * Mitochondria have4 COMPARTMENTS
2 * Mitochondria have their OWN GENETIC MATERIAL known as mtDNA
- and their OWN MACHINERY FOR ITS EXPRESSION
ROLE OF MITOCHONDRIA…7
- Regulate cell death,
- ion homeostasis,
- fat and carbohydrate metabolism,
- hormone and steroid production,
- ROS defence and production,
- cell signalling
- redox regulation.
Energy is produced by oxidative phosphorylation…6
- During oxidative phosphorylation, electrons derived from NADH and FADH2 combine with O2,
- and the energy released from these oxidation/ reduction reactions is used to drive the synthesis of ATP from ADP.
- 2H+ 2e+ + 1/2O2 —> H2O + Energy …in cytoplasm
- pmf = Dy DpH
- Isolated mitos:
Dy = 180-200 mV - Cells and in vivo:
Dy = 130-150 mV
The general organisation of a mitochondrion…6 …4 particular structures
- In the liver, an estimated 67% of the total mitochondrial protein is located in the matrix, 21% is located in the inner membrane, 6% in the outer membrane, and 6% in the intermembrane space.
- Each of these 4 regions contains a special set of proteins that mediate distinct functions.
- Cristae = are folds in the inner mitochondrial membrane.
- Outer membrane:
- separates the intermembrane space from the cytosol.
- The whole exchange of metabolites, cations and information between mitochondria and the cell occurs through the outer membrane. - Inner Membrane:
- is the active site for the electron transport chain and ATP production.
- Its integrity is crucial for mitochondrial function and depends on the supply of proteins and phospholipids. - Matrix:
- has several functions. It is where the citric acid cycle takes place.
- This is an important step in cellular respiration, which produces energy molecules called ATP.
- It contains the mitochondrial DNA in a structure called a nucleoid.
Mammalian mitochondrial DNA: 12
1 * maternally inherited
2 * 2-10 copies per mitochondrion
3 * 1,000-100,000 copies per cell
4 * compact genome, virtually no non-coding
sequences within genes, 16569 kb in size
5 * one of the smallest mitochondrial
genomes
6 * punctuated order of genes
7 * exclusively encodes membrane proteins
- circular and double stranded
9 * reduced rRNA content but sufficient for
mitoribosome assembly and function
10 * full complement of tRNAs necessary for translation
11 * D-loop contains regulatory elements for transcription
and replication
12 * The D-loop of mtDNA can be used as a unique feature to define phylogenetic relationships between species or define breeding populations
Mammalian mitochondrial DNA…
RNAs, mRNAs, tRNAs numbers
2 rRNAs (12S and 16S rRNA)
11 mRNAs
22 tRNAs
Mitochondrial mRNAs: 14
1 *“naked” start codons
2 *5’ caps
3 *no introns
4 *Most lack 5’ and 3’ UTRs, Shine-Dalgarno sequences
5 *2 mRNAs are bicistronic
* ATP8/6
* ND4L/4
6 10 mRNAs have poly(A) tails:
….7 Stabilize mRNAs in animals
…8 * Complete their stop codon
…9 * Length of tails is variable
10 *translated by mitochondrial ribosomes
11*Non-universal genetic code:
…12 * AUA is a start (instead of Ile)
…13 * UGA is Trp (not stop)
…14 * AGA and AGG are stop (not Arg)
Mitochondrial mRNAs: Diagram
slide 14
Mitochondrial DNA Transcription = 5
- Mitochondrial DNA
—- in mitochondrial matrix —-
2.POLRMT and Transcription machinery
- TRANSCRIPTION
- PROCESSING: ….POLYCISTRONIC TRANSCRIPT
- RNase P ans RNase Z - MATURATION: RNA transcripts
Mitochondrial DNA …RIBOSOME ASSEMBLY..3
- RIBOSOME ASSEMBLY
- 28S …39S- MRPL45 - mRNA MODIFICATION
- tRNA MODIFICATION
Mitochondrial DNA …TRANSLATION 4
- TRANSLATION
- RESPIRATORY COMPLEXES
- PROTEIN IMPORT ..in mitochondrion
- Then to NUCLEUS (passed from mitochondrion - cytoplasm - nucleus)
LOOK AT MITOCHONDRIAL DNA …TRANSCRIPTION, TRANSLATION, PROTEIN IMPORT ….
SLIDE 18.. UNDERSTAND AND REMEMBER THE PROCESS
The mitochondrial genetic blueprint EXPLAIN THE PROCESS…7
- Protein dna INTERACTIONS
- —-> DNase -I cleavage sequencing
- CLEAVAGE AND PROCESSING
- —> PARE sequencing
- rRNA, tRNA, mRNA
- GENE EXPRESSION
- RNA sequencing
Mitochondrial genome
Using next generation technologies we have identified that:…4
1 * The mitochondrial genome is ORGANISED AND STRUCTURED
2 * The mitochondrial GENETIC INSTRUCTIONS (RNAs)ARE MORE COMPLEX than previously thought
3 * MITOCHONDRIAL GENE EXPRESSION IS DIRECTLY LINKED TO ENERGY METABOLISM
4 * New RNAs and MODES OF GENE REGULATION
The human mitochondrial transcriptome….
Mitochondrial mRNAs: 3
1 * There is variation in the abundance of mt-mRNA levels between different tissues and within individual tissues
2 * Make up at least 30% of total RNA in the heart
3 * The stability and half-life of mt-mRNAs is different within cells
Mitochondrial ribosome and protein synthesis
Unique structure: 8
1
- 2 rRNAs (12S and 16S rRNA)
2
- 28 proteins in the small subunit
3
- 50 proteins in the large subunit
4 * reduced rRNA content
5 * increased protein content
6 * mitochondria specific ribosomal proteins
7 * recognise “naked” start codons
8 * exclusively translate membrane proteins
Mitochondrial ribosome and protein synthesis…6
- E.coli ribosome
- Yeast mitochondrial ribosome
- mammalian mitochondrial ribosome
- initiation
- elongation
- LOOK AND DIAGRAMS AND UNDERSTAND IN SLIDE 22
Unique features of mt-tRNAs: 6
1 * full complement required for translation of the 13 polypeptides, no need for additional tRNA import in mammalian mitochondria
2 * 60 sense codons for 22 tRNAs
3 * different classes of tRNAs
4 * Modified nucleotides
5 * Wobble bases
6 * mutations in tRNA genes cause defects in protein. synthesis and lead to disease
LOOK AND UNDERSTAND THE TYPES OF mt-tRNAs
Type 0
Type I
Type II
Type III
SLIDE 23
Mitochondrial tRNA modifications…
Unique features of mt-tRNAs: 3 and the types
1
*16 species of modified nucleosides
2
3 mitochondria-specific modifications:
——- 5-formylcytidine (f 5 C)
——-* 5-taurinomethyluridine (τm5 U)
——* 5- taurinomethyl-2-thiouridine (τm5 s2 U)
3
*f 5 C is required to recognize the non-universal AUA codon in addition to the AUG codon
Unique features of mt-tRNAs: LOOK AND UNDERSTAND THE Mitochondrial tRNA modifications…
DIAGRAMS
SLIDE 24
Mitochondrial rRNAs…The mitoribosome is: 5
1 * a molecular machine composed of mitochondrial ribosomes and proteins
2 * Mt-rRNA has reduced in size and has been replaced by additional proteins
3 * 2 rRNAs: 12S and 16S that form the small 28S ribosomal subunit and the large 39S ribosomal subunit
4 * TF1BM and NSUN4 methylate the 12S rRNA
5 * A tRNA is also a structural part of the large ribosomal subunit
Mitochondrial sRNAs and half-tRNAs
UNDERSTAND SLIDE 26
Mitochondrial rRNAs
The mitoribosome is:
SLIDE 25 DIAGRAM
Mitochondrial sRNAs and half-tRNAs
SLIDE 27 UNDERSTAND THE DIAGRAMS
Mitochondrial long non-coding RNAs…
LONG NONCODING RNAs DIAGRAM IN SLIDE 28***IMPORTANT
UNDERSTANDING Mitochondrial long non-coding RNAs: 3
The mitochondrial long non-coding RNAs:
1 * Highly abundant
2 * Form duplexes
3 * Produced by RNase P
Mitochondrial long non-coding RNAs DIAGRAM
LOOK AT DIAGRAM IN SLIDE 29
Regulation of mitochondrial gene expression:
Mitochondrial gene expression is regulated
by RNA-binding proteins at the level of: 4
1 * Transcription
2 * Processing
3 * Maturation
4 * Translation
Regulation of mitochondrial gene expression:
DIAGRAM
SLIDE 30 …PROCESS DIAGRAM
Summary: 4
1 * Differential regulation of mitochondrial gene expression can cause mitochondrial dysfunction with varying severities that results in a range of different disorders including, metabolic disease, cardiomyopathies, neurodegeneration, diabetes, cancer and ageing.
2 * Tissues have differential response to mitochondrial dysfunction.
3 * Mutations in either mitochondrial or nuclear genes coding for mitochondrial proteins or
RNAs can cause devastating diseases for which there is no cure nor effective treatments to date.
4 * To understand disease pathology and potential treatments, fundamental discoveries in
mitochondrial biology are necessary.
