1. Mitochondrial Genome

2. Introduction membrane-bound organelle Found only in eukaryotes Contains a small DNA circle All eukaryotic cells either have mitochondria or have nuclear genes that seem to have been derived from mitochondria. Each cell contains hundreds to thousands of mitochondria. Site of Krebs cycle and oxidative phosphorylation (the electron transport chain, or respiratory chain). two membranes: outer and inner. Folds of the inner membrane, where most of oxidative phosphorylation occurs, are called cristae. Inside inner membrane = matrix (site of Krebs cycle) Between membranes = intermembrane space Mitochondrial DNA is in the matrix, inside the inner membrane.

3. Endosymbiont Theory endosymbiont theory: The mitochondria (and also plant chloroplasts) originated from an intracellular symbiosis between 2 different free-living organisms, which got inside a primitive eukaryotic cell and developed a mutually beneficial relationship. –originally proposed in 1883 by Andreas Schimper, but extended by Lynn Margulis in 1967. –Also called symbiogenesis. Thought to have occurred about 1.5 billion years ago, at about the same time that eukaryotes originated: maybe the fundamental event in the origin of eukaryotes. Mitochondrial ribosomal RNA genes and other genes show that the original organism was in the alpha-proteobacteria family (similar to nitrogen-fixing bacteria)

5. More Endosymbiont Theory Evidence: –mitochondria have their own DNA (circular) –the inner membrane is more similar to prokaryotic membranes than to eukaryotic. By the hypothesis, the inner membrane was the original prokaryotic membrane and the outer membrane was from the primitive eukaryote that swallowed it. –mitochondria make their own ribosomes, which are of the prokaryotic 70S type, not the eukaryotic 80S type. –mitochondria are sensitive to many bacterial inhibitors that don’t affect the rest of the eukaryotic cell, such as streptomycin, chloramphenicol, rifampicin. –Also, inhibitors of eukaryotic protein synthesis don’t affect mitochondrial protein synthesis. –mitochondrial protein synthesis starts with N-formyl methionine, as in the bacteria but unlike eukaryotes. Most of the original bacterial genes have migrated into the nucleus. Mechanism unclear: Eukaryotes that lack mitochondria generally have some mitochondrial genes in their nucleus, evidence that their ancestors had mitochondria that were lost during evolution. –The origin of eukaryotes vs. origin of mitochondria are not clearly distinct events.

6. Phylogeny of Mitochondrial DNA

7. Endosymbiont Hypothesis This is actually a secondary endosymbiosis: the largest cell is engulfing a photosynthetic eukaryote, which already contains chloroplasts.

8. Mitochondrial Function Krebs cycle: –Pyruvate, the product of glycolysis, is produced in the cytoplasm. –It is transported into the mitochondrial matrix (inside the inner membrane). –There, it is converted into acetyl CoA. –Fatty acids, from the breakdown of lipids, are also transported into the matrix and converted to acetyl CoA. –The Krebs cycle then converts acetyl CoA into carbon dioxide and high energy electrons. The high energy electrons are carried by NADH and FADH 2. Electron Transport: –The high energy electrons are removed from NADH and FADH 2, and passed through three protein complexes embedded in the inner membrane. –Each complex uses some of the electrons’ energy to pump H + ions out of the matrix into the intermembrane space. –The final protein complex gives the electrons to oxygen, converting it to water. –The H + ions come back into the matrix, down the concentration gradient, through a fourth complex, ATP synthase (also called ATPase), which uses their energy to generate ATP from ADP and inorganic phosphate. In brown fat, the synthesis of ATP is uncoupled from the flow of H + ions back into the matrix. The H + ions flow through a protein called thermogenin, and not through the ATPase. The energy is converted into heat: the primary way we keep warm in cold weather.

9. Mitochondrial Function

11. Genome Structure The mitochondrial genome is a circle, 16.6 kb of DNA. A typical bacterial genome is 2-4 Mbp. The two strands are notably different in base composition, leading to one strand being “heavy” (the H strand) and the other light (the L strand). Both strands encode genes, although more are on the H strand. A short region (1121 bp), the D loop (D = “displacement”), is a DNA triple helix: there are 2 overlapping copies of the H strand there. The D loop is also the site where most of replication and transcription is controlled. Genes are tightly packed, with almost no non-coding DNA outside of the D loop. In one case, two genes overlap: they share 43 bp, using different reading frames. Human mitochondrial genes contain no introns, although introns are found in the mitochondria of other groups (plants, for instance).

12. Mitochondrial Genes Genes: total of 37. 22 transfer RNAs, 2 ribosomal RNAs, 13 polypeptides. Transfer RNA: only 60 of the 64 codons code for amino acids. –Total of 22 tRNAs: 8 tRNAs cover all 4 3 rd base positions with the same amino acid, and the remaining 14 tRNAs each cover two 3 rd base positions (purines or pyrimidines). Thus, all 60 codons are covered. Ribosomal RNA: 16S and 23S, which are standard sizes for bacterial rRNAs. –Bacterial ribosomes don’t use 5S or 5.8S rRNAs. polypeptides: all are components of the electron transport chain. Other components are encoded in the nucleus and transported to the mitochondria after translation.

13. Genetic Code The mitochondrial genetic code has drifted from the universal code: there are so few polypeptides that changes in the code are tolerated. Human mitochondrial code is different from other groups such as plants or fungi. Uses 2 of the 3 universal stop codons, but also uses 2 other codons as stop codons. Also, UGA codes for tryptophan in the mitochondrial, while it is a stop codon in the universal code. AUA gives methionine in the mitochondria instead of isoleucine.

14. Replication Replication starts with the H strand. The origin of replication for the H strand (OriH) is in the D loop, and it is initiated by an RNA primer generated from the L strand transcript. After the new H strand is about 2/3 complete, the L strand origin of replication (OriL) is uncovered. The L strand origin is on the old H strand; it is “uncovered” when the old H strand is displaced by the DNA polymerase synthesizing the new H strand. The L strand origin folds into a stem-loop structure, which acts as a primer, and replication of the L strand begins. Replication can be said to be bidirectional but asynchronous, unlike replication of nuclear DNA, which proceeds in both directions simultaneously.

15. Transcription and RNA Processing Both strands are transcribed as single RNA molecules The D loop contains one promoter for each strand, and the entire strand is transcribed. The RNA is then cut into individual RNAs for each gene. This involves at least 43 separate cleavages. Protein-coding genes are given poly-A tails. –No 5’ cap (which is found for nuclear transcripts) –No introns –Many mitochondrial transcripts have no 5’ UTR or 3’UTR (untranslated regions): translation goes from the first base of the mRNA to the last base. –The first A of the poly A tail is the last base of the stop codon (UGA or UAA) for many transcripts! rRNA and tRNA molecules are modified as necessary. –Transfer RNA molecules contain many modified bases RNA editing also occurs: enzymes that convert specific C’s to U’s by deamination –In plants, RNA editing in the mitochondria occurs at many sites Mitochondrial RNA polymerase is coded in the nucleus, but it is not similar to either eukaryotic or prokaryotic RNA polymerases. –It seems to be a modified version of a bacteriophage RNA polymerase. It has just 1 subunit, where typical eukaryotic RNA polymerases have 10-123 subunits and prokaryotic RNA polymerases have 5 subunits

16. Transcription Map Heavy strand genes are in the outer circle; light strand gens in the inner circle. There are 2 heavy strand promoters: H1 and H2, and a light strand promoter: LSP. H1 makes a short transcript for ribosomal RNA only. Black boxes are protein-coding genes; hatched boxes are ribosomal RNA genes; circles are transfer RNA genes.

17. RNA Editing RNA editing is a fairly rare form of RNA processing, in which nucleotides are added, removed, or altered in the RNA sequence after transcription has occurred. –RNA editing has been seen in all domains of life, and in many different types of RNA, including protein-coding messenger RNA. One simple mechanism: an enzyme deaminates a specific C in a mRNA, converting it to U. In DNA this would be repaired, but U is a legitimate RNA base. –In apolioprotein, this change converts a CAA (glutamine) codon into a UAA stop codon, which allows translation of a much shorter protein. –Another deamination, from adenine to inosine, is common in mammals. Inosine acts like guanine in translation and in base pairing.

18. Transporting Proteins into the Mitochondria Nuclear-coded proteins are transported across the outer membrane by the translocase of the outer membrane (TOM) complex, a multi-subunit integral membrane protein. If necessary, proteins are transported into or through the inner membrane by the translocase of the inner membrane (TIM) protein complexes, which has 2 independent components (TIM22 and TIM23), one for inner membrane proteins and one for matrix proteins Proteins destined for the matrix or inner membrane have an N-terminal signal sequence: –Very similar in function to signal sequence for endoplasmic reticulum, except the mitochondrial proteins are completely synthesized first, then transported. ER proteins are synthesized directly into or through the membrane. –The signal sequence is an amphipathic alpha helix: charged on one side and hydrophobic on the other side. –Recognized by components of TOM and TIM Proteins destined for other locations have internal signal sequences (not N-terminal)

19. Mitochondrial Protein Transport

20. Mitochondrial Genetics Maternal inheritance: Inherited through the mother (egg) only. Allows tracing female line back in time. A few sperm mitochondria enter the egg, but they are degraded and lost. Mutation rate in mtDNA is very high: 10 times the nuclear rate. mtDNA is associated with the inner membrane, the site of oxidative phosphorylation. Oxidative phosphorylation generates large amounts of “reactive oxygen species” (peroxide and superoxide), which are quite mutagenic.

21. Heteroplasmy Sometimes an individual has more than one kind of mitochondria. This is called heteroplasmy. Since mitochondria are divided randomly during cell division, different cells get different proportions of the two types. If one mitochondrial type is mutant and the other is normal, severity of symptoms will vary in different tissues depending on the proportions of the two types. Inheritance: During oogenesis (egg formation), random segregation of the two types can lead to some offspring inheriting a mitochondrial disease while other offspring are normal. –No simple segregation ratio: a sign of a mitochondrial disease –Severity of symptoms varies between offspring –Oocytes contain only about 10 mitochondria

22. Genetic Diseases in general: malfunctions of respiratory chain affect high metabolism tissues the most: nervous system, muscles, kidney, liver. Heteroplasmy is very common, meaning that symptoms vary widely and affect different tissues at different rates Mutations in many different genes can produce similar diseases

23. Leber's Hereditary Optic Neuropathy Progressive loss of vision, from central to peripheral, usually beginning in 20's. Eyes can be affected several months apart, or simultaneously. About 85% are male (no good reason why). Recurrence risk for siblings around 20% (heteroplasmy); many spontaneous cases. Due to death of optic nerve fibers. Most due to change in conserved Arg to His in NADH dehydrogenase, but 18 total mutations known, all missense in respiratory chain.

24. MERRF Myoclonic epilepsy and ragged red fiber disease (MERRF). CNS symptoms: epilepsy, deafness, dementia. Muscle twitching and weakness. Skeletal and heart muscles appear abnormal, mitochondria appear abnormal. Multiple enzyme defects in respiratory chain. Ragged red fibers are clumps of abnormal mitochondria that accumulate in the muscle fibers that stain red using a standard histological stain. Most (80%) MERRF cases are due to A --> G mutation in lysine tRNA (mutation A8344G). Easy to assay for; CviJI restriction site is altered. –Some cases involve other tRNA genes Lots of variation in inheritance of disease, due to heteroplasmy. Good correlation between % mutant mitochondria and disease severity.

25. Kearns-Sayre Syndrome Symptoms: paralysis of the muscles, droopy eyelids, retinal degeneration, cardiac muscle problems, seizures, many other symptoms irregularly. Cause: Large deletions of mtDNA in the protein coding gene region. This leads to a failure of oxidative phosphorylation, thus low energy production in the cell. Heteroplasmy necessary for survival. Mostly spontaneous--rarely passed to offspring. Many variants.

26. Mitochondria, Free Radicals, and Aging One theory of aging: DNA and other biological molecules are damaged by oxidation. Damage is caused by reactive oxygen species (ROS): molecules containing oxygen that act as strong oxidizing agents. –free radicals: molecules that have an unpaired electron. Examples: superoxide: O 2 - and nitric oxide (NO). They attack other molecules by pulling off an electron (oxidation), creating new free radicals. –Also bad: peroxide Damage: crosslinking of DNA, oxidation of lipids and proteins –Mitochondria that have too much oxidative damage induce apoptosis (programmed cell death) ROS are created by ionizing radiation and energetic chemical reactions (cigarette smoking, for example). Also created in the mitochondria as a byproduct of the electron transport chain. Usually, the last step involves reducing an O 2 molecule to water by donating a pair of electrons. However, sometimes only a single electron gets donated, producing superoxide instead of water.

27. Dealing With ROS We have enzyme systems to remove superoxide, peroxide, and other ROS: –Superoxide dismutase converts superoxide to peroxide –Catalase converts peroxide to water Also: anti-oxidants (which have extra electrons to donate to the free radicals) remove ROS

28. More Aging The theory: mitochondrial DNA mutated by ROS produces defective electron transport proteins, which in turn generate more free radicals: positive feedback. –ROS-stressed mitochondria induce apoptosis –The free radicals oxidize lipids and proteins in the cells –Causes wrinkled skin, cancer, arterial plaque formation, many other chronic age-related diseases

29. Three Parent In Vitro Fertilization The idea is to use the nucleus from one woman and the egg cytoplasm (especially the mitochondria) from another woman. –This allows woman with defective mitochondrial DNA to produce healthy children. Procedure: remove the nucleus from a healthy egg, then transfer in the nucleus from an egg with bad mitochondria. Then, fertilize in vitro with a sperm nucleus. Has been performed in Great Britain, but currently banned in the US. It is a form of gene therapy that affects future generations; such procedures are viewed cautiously.