BIOL21101_Module13_organelles_3_

Heteroplasmy

The presence of both normal and mutated mitochondrial DNA within a single cell.

Mitochondrial DNA (mtDNA)

The circular DNA located in mitochondria that is inherited maternally and codes for polypeptides in the respiratory chain.

RNA Polymerase

An enzyme that synthesizes RNA from a DNA template during the process of transcription.

MitoTracker

A dye used to label mitochondria in live cells to study their dynamics and distribution. MitoTracker is a lipophilic cationic dye, meaning it has a positive charge and can cross lipid membranes. Mitochondria have a negative membrane potential across their inner mitochondrial membrane, which drives the accumulation of the dye specifically into the mitochondria.

Mechanism: The negatively charged mitochondrial matrix attracts the positively charged dye.This allows for selective staining of mitochondria, enabling visualization and tracking of mitochondrial behavior in various cellular processes.

RNase P

A ribonucleoprotein that catalyzes the maturation of the 5′ end of tRNA.

RNase Z

A protein responsible for the maturation of the 3′ end of tRNA.

Mitochondrial Disorders

Genetic disorders caused by dysfunctional mitochondria affecting energy production.

MELAS syndrome

The central issue in MELAS syndrome is the mutation in the MT-TL1 gene, which encodes the mitochondrial tRNA for leucine (tRNA-Leu(UUR)). This mutation disrupts mitochondrial protein synthesis, ultimately leading to impaired energy production. A mitochondrial disorder characterized by mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes.

MERRF syndrome

A mitochondrial disorder characterized by myoclonus epilepsy with ragged-red fibers. mutation in the MT-TK gene, which encodes the mitochondrial tRNA for lysine (tRNA-Lys). this causes improper folding and instability of tRNA-Lys so incapability of providing lysine during mtRNA translation leading to neurological and muscular symptoms.

LHON syndrome

Leber hereditary optic neuropathy, a mitochondrial disorder leading to vision loss. Mutations in MT-ND4, MT-ND1, and MT-ND6 impair the activity of Complex I, which is responsible for transferring electrons from NADH to ubiquinone (coenzyme Q10).

• This disruption leads to:

  • Reduced ATP production.

  • Accumulation of electrons, increasing reactive oxygen species (ROS) production.

• Oxidative Stress:

  • Elevated ROS damages mitochondrial and cellular components, including lipids, proteins, and mtDNA.

  • Optic nerve cells, which have high metabolic demands, are particularly vulnerable to oxidative damage.

currently no definitve cure

Reactive Oxygen Species (ROS)

Chemically reactive molecules containing oxygen that can lead to cellular damage. Conditions like MELAS and LHON involve excessive ROS production due to defective electron transport chain complexes.

Mutation Rate

The frequency at which mutations occur in a given length of DNA over a specific time frame.

Chloroplast DNA (cpDNA)

Genetic material in chloroplasts, usually maternally inherited, pivotal in photosynthesis.

Polycistronic Transcripts

prokaryotic characteristic found in mt and cp due to their origins. RNA molecules that encode multiple proteins from a single ribosomal RNA gene.

Zinc-Finger Nucleases (ZFNs)

Tools for targeted genetic modifications that utilize zinc-finger proteins to bind DNA sequences.

ZFNs specific to mtDNA sequences allow for selective degradation of mutated mtDNA, shifting heteroplasmy toward the wild-type genome. For example:

• Therapeutic applications: Used to target mitochondrial diseases like MELAS or LHON.

Transcription Activator-Like Effector Nucleases (TALENs)

Customized nucleases used for genome editing, based on DNA-binding proteins. Based on Transcription Activator-Like Effectors (TALEs), originally derived from bacteria of the genus Xanthomonas.

• TALENs bind to specific DNA sequences via their customized TALE repeats.

• Two TALENs are designed to flank the target site, each binding one strand of the DNA.This allows for precise double-strand breaks, enabling targeted modifications in the genome.

Exonucleases

Exonucleases are enzymes that cleave nucleotides one at a time from the ends of polynucleotide chains, playing vital roles in processes such as DNA repair, RNA processing, and degradation.

Endonucleases

Endonucleases are enzymes that cleave the phosphodiester bonds within polynucleotide chains, effectively cutting DNA or RNA at specific internal sites. This function is essential for various cellular mechanisms, including DNA repair, recombination, and RNA processing

Polyadenylation

The addition of a poly(A) tail to a RNA molecule, which can affect its stability and translation.

In chloroplasts it is done by Ntr-PAP or PNPase to trigger cpRNA degradation (that the same enzymes perform as they have exonucleotic activity)

in mitochondria, it is done post-transcriptionally by mt-PAP to complete missing STOP codons in mitochondrial transcripts, poly-A is shorter than for us. Helps with stability and enhances translation

Many mitochondrial mRNAs are encoded without full stop codons in the mitochondrial genome. For example:

• The mRNA may end with "U" or "UA" instead of a complete "UAA" or "UGA".

Ribonuclease (RNAses)

An enzyme that catalyzes the degradation of RNA into smaller components. there are several in the genome, have mitochondria, nucleus and chloroplast specific ones, they are involved in processing tRNA in nucleus. In mt and cp, same catalytic reaction

Mutation Accumulation

The gradual accumulation of mutations over generations within a population.

Gene Expression

The process through which information from a gene is used to synthesize functional gene products.

Gene Transfer

The process of moving genetic material from one organism or cell to another.

Circular DNA

DNA molecules that have no free ends, forming a closed loop, commonly seen in bacteria and mitochondria.

Copy Number Variants

Sections of the genome that have been duplicated or deleted, leading to variations in the number of copies of a particular gene.

Polymerase Chain Reaction (PCR)

A technique used to amplify DNA sequences, making millions of copies from a small initial sample.

Antisense RNA

A strand of RNA complementary to a sense strand, used in gene regulation.

D-loop

A displacement loop in mtDNA critical in initiation of replication. this area has a high mutation rate compared to the rest of mtDNA. forms when leading mrDNA strand begins at the origin of replication and displaces the complementary (light) strand, creating a loop structure.

Endosymbiotic Theory

The endosymbiotic theory posits that eukaryotic cells evolved from prokaryotic cells that engulfed one another. This explains how complex life forms, like plants and animals, emerged from simpler bacterial ancestors. For example, mitochondria and chloroplasts, which are critical for energy production in eukaryotes, resemble bacteria in their DNA structure and replication processes, suggesting they were once independent organisms.

α-proteobacteria/aerobic prokaryote for mitochondria and cyanobacteria for chloroplasts

Heteroplasmy

Heteroplasmy refers to the coexistence of both normal and mutated mitochondrial DNA within a cell. This phenomenon is significant because it can influence the severity of mitochondrial diseases; for instance, if a muscle cell has a higher proportion of mutated mtDNA compared to normal mtDNA, it may lead to energy deficits affecting muscle function.

Mitochondrial DNA (mtDNA)

Mitochondrial DNA is a circular DNA found in mitochondria, distinct from nuclear DNA, and it is inherited solely from the mother. It encodes 37 genes that are essential for mitochondrial function, particularly related to oxidative phosphorylation. The maternal inheritance pattern is crucial because it means that mutations can be traced through matrilineal lines, providing insights into evolutionary biology and ancestry.

Mitochondrial Disorders

Mitochondrial disorders are a group of genetic conditions caused by dysfunctional mitochondria, impairing energy production in cells. Common examples include MELAS syndrome, which presents with neurological symptoms due to energy deficits in brain cells, and LHON syndrome, characterized by vision loss due to preferential vulnerability of retinal ganglion cells to energy failure.

Reactive Oxygen Species (ROS)

Reactive Oxygen Species are highly reactive molecules derived from oxygen that can cause oxidative stress, leading to cellular damage. They are produced in normal metabolic processes but can be generated in excess during stress or disease conditions. For example, excessive ROS can damage mitochondrial DNA, contributing to aging and the development of various diseases, including cancer.

Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) is a widely-used technique to amplify specific DNA sequences, creating millions of copies from a tiny initial sample. This process involves repeated cycles of denaturation, annealing, and extension, allowing researchers to analyze DNA for various applications, such as genetic testing, forensics, and infectious disease diagnosis.

Gene Expression

Gene expression is the process by which the information encoded in a gene is translated into functional products, typically proteins. This involves transcription of DNA into mRNA and subsequent translation of mRNA into protein. The regulation of gene expression is crucial for cell function and differentiation, and understanding this process is fundamental in fields like developmental biology and genetics.

Codon

A codon is a sequence of three nucleotides in mRNA that corresponds to a specific amino acid or a stop signal during protein synthesis. For example, the codon AUG encodes for methionine and serves as the start signal for translation. The redundancy in the genetic code means that multiple codons can code for the same amino acid, influencing genetic mutations and protein variations.

Exonucleases

Exonucleases are enzymes that remove nucleotide units one at a time from the ends of a polynucleotide chain. They play vital roles in DNA repair and degradation processes. For instance, in DNA replication, exonucleases help to ensure fidelity by removing incorrectly incorporated nucleotides, thus safeguarding genetic information.

Antisense RNA

Antisense RNA is a strand of RNA that is complementary to a sense strand of mRNA, and it is used in cellular mechanisms to regulate gene expression. For example, in gene silencing technologies, antisense RNA can bind to target mRNA, preventing its translation into proteins, thereby offering therapeutic potential in conditions caused by gene overexpression.

tRNA Maturation Pathway

1. Precursor tRNA is transcribed by RNA polymerase III.

2. RNase P removes the 5' leader sequence.

3. RNase Z cleaves the 3' trailer sequence.

4. The CCA sequence is added at the 3' end (if not already encoded in the gene).

5. The mature tRNA undergoes other modifications (e.g., methylation, pseudouridylation) to become fully functional.

tRNA Processing

22 encoding tRNA in mitochondria, 30-35 in chloroplast due to the fact they encode more of their own translational machinery.

1- transcription of precursor tRNA by RNA related to T7 RNAP (bacteriophage pol) for mitochondria= mtRNAP that will transcribe whole strand with no termination btw each gene (polycistronic)

removal of the 5' leader sequence by RNase P, and cleavage of the 3' trailer sequence by RNase Z. Following this, the addition of a CCA sequence at the 3' end occurs if it is not already encoded in the DNA. Finally, further modifications such as methylation and pseudouridylation may take place to fully functionalize the mature tRNA.

hPNPase and PNPase

hPNPase (human polynucleotide phosphorylase) is a human enzyme involved in RNA processing and degradation, often associated with mitochondrial functions. PNPase (polynucleotide phosphorylase) refers to a similar enzyme found in bacteria and mitochondria that also contributes to RNA processing but can differ in specific functionalities and regulatory mechanisms depending on the organism.

hSuv3

hSuv3 is a mitochondrial RNA helicase that plays a critical role in the processing and degradation of mitochondrial RNA. It is involved in maintaining mitochondrial RNA homeostasis and facilitating the unwinding of RNA structures during various RNA metabolic processes.

Codon Usage in cpDNA vs mtDNA

The triplet code for chloroplast DNA (cpDNA) is generally the same as that of the nuclear genome, allowing for similar codon assignments across these genomes. However, mitochondrial DNA (mtDNA) has unique codon usage, such as variations in stop codons, which differ from the nuclear and chloroplast genomes, impacting the synthesis of proteins.

Bidirectionality of Transcription

Bidirectionality of transcription refers to the ability of transcription to occur in both directions along a DNA strand. In eukaryotic cells, this is evident in mitochondrial and nuclear genomes where certain genes may be transcribed from both strands, allowing for complex regulation of gene expression.