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mitochondrial genome.pptx

  1. Mitochondrial genome
  2. • Mitochondria are dynamic organelles that are present in almost all eukaryotic cells and play a crucial role in several cellular pathways. Their most recognizable role is providing the cell with energy in the form of ATP via OxPhos. However, many other functions have been assigned to mitochondria, including the integration of metabolic pathways (such as the biosyntheses of heme, iron–sulfur clusters, and nucleotides), apoptosis, and reactive oxidative species (ROS) signaling
  3. • The endosymbiotic theory proposes that mitochondria originated as free-living Alphaproteobacteria that were internalized by a pre- eukaryotic host cell, leading to the formation of the modern eukaryotic cell. In the course of evolution, the genome of the original alphaproteobacterial symbiont has undergone extensive reduction. The majority of its genes have either been lost, owing to redundancy, or transferred to the host nuclear genome. Furthermore, mitochondria have lost autonomy over their genome maintenance and expression to the host cell. Nonetheless, in almost all cases, eukaryotic mitochondria retain a minimal genome, of variable size and gene content, that is present in many copies within their matrix.
  4. • Human mitochondrial DNA (mtDNA) is a circular molecule of 16.5 kb which encodes a small subset of the structural polypeptide components required for OxPhos. These mRNAs aretranscribed and then translated within the mitochondrial matrix by a dedicated, unique, and highly specialized machinery. The RNA components of the mitochondrial gene expression system, two mitochondrial ribosomal RNAs (mt-rRNAs) and 22 mt-tRNAs, are also encoded by mtDNA, whereas all other protein components are encoded by nuclear genes and imported into mitochondria from the cytosol
  5. • 250–300 nucleus-encoded proteins are dedicated to serve mitochondrial gene expression. This includes RNA polymerase and transcription factors, endonucleases for RNA precursor processing, aminoacyl-tRNA synthetases, RNA- modifying enzymes, the structural components and biogenesis factors for the mitochondrial ribosome, translation factors, and other auxiliary factors
  6. • Map of the human mitochondrial genome (16 569 bp). The outer circle represents the H-strand, containing the majority of the genes; the inner circle represents the L-strand. The D-loop is shown as a three-stranded structure. The origins of H-strand (OH) and L-strand (OL) replication and the direction of DNA synthesis are indicated by long bent arrows; the initiation of transcription sites (ITL, ITH1, ITH2) and the direction of RNA synthesis are denoted by short bent arrows. The binding site for the mitochondrial transcription terminator (mtTERM) is indicated. The 22 tRNA genes are depicted by dots and the single letter code of the amino acid (isoacceptors for serine and leucine are distinguished by their codon sequence). The genes coding for the two rRNA species (12S and 16S) and the 13 protein coding genes are depicted by shaded boxes. ND, CO and ATPase refer to genes coding for subunits of NADH:ubiquinone oxidoreductase, ferrocytochrome c:oxygen oxidoreductase (cytochrome c oxidase) and F1F0-ATP synthase, respectively, whereas Cyt b encodes apocytochrome b of ubiquinol:ferricytochrome c oxidoreductase.
  7. Basic Features • The strands of the DNA duplex can be distinguished on the basis of G+T base composition which results in different buoyant densities of each strand (‘heavy’ and ‘light’) in denaturing caesium chloride gradients • Most information is encoded on the heavy (H) strand, with genes for two rRNAs, 14 tRNAs, and 12 polypeptides. • The light (L) strand codes for eight tRNAs and a single polypeptide. • All 13 protein products are constituents of the enzyme complexes of the oxidative phosphorylation system • The genes lack introns and, except for one regulatory region, intergenetic sequences are absent or limited to a few bases. Both rRNA and tRNA molecules are unusually small • Some of the protein genes are overlapping and, in many cases, part of the termination codons are not encoded but are generated post- transcriptionally by polyadenylation of the mRNAs
  8. Unique Codons • mitochondrial protein sequences revealed deviations from the standard genetic code and later even variations in codon usage were found in mitochondria from different species • In mtDNA of most phylogenetic groups, TGA is used as a tryptophan codon, rather than as a termination codon. • AGR (R=A, G) specifies a stop in mtDNA of vertebrates, codes for serine in mtDNA of echinoderms and codes for arginine in mtDNA of yeast, as in the standard genetic code.
  9. mtDNA Inheritance • • mtDNA is inherited entirely from the mother • • Sperm carries the father's mtDNA in its tail, which is lost • during fertilization • • mtDNA inheritance is non-Mendelian, because • Mendelian inheritance presumes that half the genetic • material of an embryo derives from each parent • • mtDNA passes unchanged from mother to offspring by • this mechanism
  10. Characteristics of mtDNA • The two strands of mtDNA have significantly different compositions from nuclear DNA and from one another - The heavy (H) strand is rich in purines (adenine and guanine) - The light (L) strand is rich in pyrimidines (thymine and cytosine) • mtDNA is highly conserved, so it is useful for phylogenetic study • 80% of mtDNA encodes for functional mitochondrial proteins, and therefore most mtDNA mutations lead to functional anomalies • Mutations in nuclear DNA may also have a wide array of effects on mtDNA replication • mtDNA is devoid of introns mtDNA contains only a few non-coding intergenic regions • The size and number of mtDNA - Human mtDNA is 16,569 bp in length - Human cells typically contain thousands of copies of mtDNA, several copies per mitochondrion
  11. mtDNA genes mtDNA encodes for 37 genes • There are 13 peptide coding genes, including the following peptides • Transcription factor A • The mtRNA processing ribonuclease P • The transcription termination factor • The mitochondrial peptides are synthesized on mitochondrial ribosome • The heavy strand encodes the 2 rRNAs, 12 polypeptides, and 14 tRNAs • The light strand encodes 1 polypeptide, and 8 tRNAs
  12. mtDNA expression - Transcription initiation sites of mtDNA • The promoters of Hand L strands (termed PH and PL) are both located in the D-Ioop region and 150 bp apart (seemtDNA replication at the end of this section) (Figure 26) • Heavy-strand transcription starts at nucleotide 561 • Light-strand transcription starts at nucleotide 407
  13. Transcription of mtDNAstarts from the promoters in the D-Ioop region and continues in opposing directions for the two strands around the circle to generate large multigenic transcripts • Transcription initiation in mitochondria involves three types of proteins • The mtRNApolymerase (POLRMT) • Mitochondrial transcription factorA (TFAM) • Mitochondrial transcription factors BI and B2 (TFBIM, TFB2M) • POLRMT, TFAM,and TFBIMor TFB2M assemble at the mitochondrial promoters and begin transcription
  14. Promoters of mitochondrial gene expression • Heavy strand I (HI) promoters initiate transcription of the entire heavy strand • Heavy strand 2 (H2) promoters initiate transcription of the two mitochondrialrRNAs • Light strand (L) promoter initiates transcripts of the entire light strand
  15. Mitochondrial mRNA - Mitochondrial mRNAs are small molecules - Full-length transcripts are cut into functional tRNA, rRNA, and mRNAmolecules - Mitochondrial mRNAlacks a 5' cap structure - Mitochondrial mRNAslack both a 5' and a 3' UTR • - The first codon specifies N-formylmethionine and is located at or very near the 5' end
  16. Regulation of mtDNAexpression - mtDNA expressiondepends on a large number of proteins encoded by nuclearDNA - The regulatory proteins are synthesized in the cytosol and enter the mitochondria via specialized pores - mtDNA replication is regulated by only one regulatory region controlling both the heavy and the light strands
  17. Post-transcriptional modification of mitochondrial mRNA - Mitochondrial mRNAs are processed by mitochondrial ribonuclease (mtRNase)cleavage of the transcript - The light strand may produce either short transcripts, which serve as primers for mtDNAreplication, or a long transcript for peptide and tRNAproduction - The productionof primer occurs by processing of light- strand transcripts with the mtRNAseP - The Hand L strand mRNAmolecules are polyadenylated by a mitochondrial poly(A) polymerase, imported from the cytosol
  18. Mitochondrial mRNA translation - The protein components necessary for mitochondrial translation, including ribosomal proteins, tRNA synthetases, ribonucleases, initiation, and elongation factors, are all encoded by nuclear genes. - Mitochondrial protein synthesis and DNA replication are thus under nuclear regulatory control - Mitochondrial translation is bacteria-like both in its sensitivity to antibiotics that act on the ribosome, and in its use of N- formylmethionyl-tRNA for initiation - Mitochondrial ribosomes are smaller than those found in the cytosol, and have a sedimentation coefficient of 55 S instead of the denser 80 S sedimentation coefficient for cytosolic ribosomes or 70 S coefficient for bacterial ribosomes
  19. mtDNA Replication • mtDNA replication is an asynchronou s process, which begins at the origin of the H strand mtDNA replication is controlled by chromosomes in the nucleus based on how many mitochondria the particular cell needs at that time - When the replication apparatu s meets the origin of the L strand, it is forced into a single-strand configuration by the extending daughter H strand, and L-strand replication begins at this point RNA derived from the L-strand promoter serves as a primer for H- strand DNA replication.
  20. The D-Ioop (displacement loop) is a I I23-ba se stretch of DNA, often triple- stranded, which contains sites for DNA-binding protein s that control mtDNA replication and transcription - The D-Ioop contains the promote rs for both the H- and L-strand transcripts - mtDNA replication causes the D-Ioop to move along the heavy strand as mtDNA polymerase-y produces a complimentary replica strand • Heavy-strand DNA replication begins at the D-Ioop and proceed s in a 5'-3' direction until returning to the origin of replication • DNA polymerase y begins in the reverse direction to produce a complimentary replica of the lightstrand when replication of the heavy strand reaches the light strand replication origin (OL) • Two identical double-strand mtDNA molecules are the result of this process • When mitochondria have enough copies of mtDNA, sufficient mitochondrial proteins, and adequate surface area, a nuclear protein may permit the mitochondrion to divide by fission into two daughter mitochondria
  21. • - mtDNA is susceptible to insult by all the same processes that damage nuclear DNA • - mtDNA is especially susceptible to insult by reactive oxygen species, which are prevalent in mitochondria • • Because mtDNA is not bound to histones, it is exposed to damage caused by free oxygen radicals produced by electron transfer during oxidative phosphorylation of the respiratory chain • • mtDNA also undergoes the same types of mutation as nuclear DNA including spontaneous modifications and replication errors • mtDNA damage
  22. mtDNA mutations - The rate of mutation in mtDNA is calculated to be about 10 times greater than that of nuclear DNA - The mtDNA mutations may be either acquired or inherited - Several different mutations of mtDNA may present clinically as the same disease - Large deletions and duplications of mtDNA increase with age • This may account for some aging processes in oxygen-dependent organs, such as brain, kidney, muscle, and heart • Mutant electron transfer proteins may release more oxygen-free radicals into the mitochondrial matrix, accelerating the aging process in some cases of Alzheimer's and coronary artery disease - There are hypervariable segments (HVI and HV2) located at base 57-372 and base 16,024-16,383, respectively. The rate of mutation in these regions is significantly higher than in the rest of mtDNA
  23. mtDNA repair - mtDNA does not code for any DNA repair proteins - Proteins from the cytosol under nuclear control enter the mitochondrion through specialized membrane pores - Recent evidence has suggested that mitochondria have enzymes to proofread mtDNA and fix mutations owing to free radicals - Evidence for nucleotide excision repair, direct damage reversal, mismatch repair, and recombinational repair mechanisms have also been found in mitochondria - As with nuclear DNA repair, the ability of mitochondria to repair DNA damage declines with age
  24. Mitochondrial Disease • Mitochondrial diseases result from failures of the mitochondrial specialized compartments for oxidative phosphorylation, which are present in every cell of the body except red blood cells - About one in 4000 children in the United States will develop a mitochondrial disease by the age of 10 years - 1000-4000 children per year in the United Sates are born with some type of congenital mitochondrial disease - Mitochondrial diseases may either be observable at birth, or symptoms may not be seen until late adulthood - Heteroplasmy refers to a phenomenon in which the number of mutant versus wild-type mitochondria varies from cell to cell and from tissue to tissue When a tissue reaches a certain ratio of mutant to wild-type mitochondria, a disease becomes manifest - Mitochondrial disease may be caused either by mtDNA mutations (acquired or inherited) or by mutations in nuclear DNA coding for mitochondrial components - Types of mutations • Homoplasmic: similar distribution of mtDNA mutation in all tissues • Heteroplasmic: variable distribution of mtDNA mutation in different cells or tissues
  25. • Typical symptoms of mitochondrial disease include - Loss of muscle coordination, muscle weakness Neurologic problems, seizures Visual and/or hearing problems Developmental delays, learning disabilities Heart, liver, or kidney disease Gastrointestinal disorders and severe constipation Diabetes Increased risk of infection Thyroid and/or adrenal dysfunction Autonomic dysfunction Neuropsychologic changes characterized by confusion, disorientation, and memory loss
  26. The diagnosis of mitochondrial disease is problematic There is no reliable and consistent means of diagnosis Evaluating the patient's family history is essential - Diagnosis may require one of the few physicians who specialize in mitochondrial disease - Diagnosis can be made by blood DNA testing and/or muscle biopsy but neither of these tests is completely reliable