Introduction Flashcards
Nucleolus
transcription of rRNA and preassembly of ribosomes
inside the nucleus
Cell cycle
G1: protein synthesis, organelle production, cell growth, longest and variable
G0: G1-arresten cells, terminally differentiated cells (muscle cells, neurons)
S: replication of chromosomes
G2: cell prepares for mitosis
M: mitosis
Mitosis
Prophase: condensation of chromosomes, spindle formation
prometaphase: nuclear envelope breaks apart
Metaphase: chromosomes line up between the spindle poles
Anaphase: separation of sister chromatids, movement to opposite poles
telophase/cytokinesis: formation of nuclear envelope, decondensation of chromosomes, cell division (cytokinesis)
Regulation of the cell cycle
a strict control of cell cycle is essential for the organism
regulated by cell cycle-specific cyclins and cyclin-dependent kinases (CDKs)
CDKs are activated by cyclins (complex) and inhibited by CDK inhibitors
CDK phosphorylates (activates) nuclear target proteins that drives the cell cycle
different cyclins are expressed/degraded in different phases of the cell cycle
Cell cycle arrest
CDK inhibitors (CKIs) arrest the cell cycle under conditions like:
- contact with other cells
- DNA damage
- Terminal cell differentiation
- senescence
CKIs are often down-regulated in cancer!!
Apoptosis
- normal physiological mechanism in multicellular organisms
- important for development (separation of fingers and toes in vertebrates=
- ca 0,1% of the cells in your body die every day by apoptosis (cell homeostasis)
- protects against cancer and other dieases (elimination of mutated or virus-infected cells)
- Abnormal apoptosis may result in neurodegenerative diseases (Alzheimer, Huntington, Parkinson) and tissue damage after stroke/heart attack
Apoptosis mechanisms - two main pathways
Extrinsic pathway: external signal, ligand binding (Fas ligand to Fas receptor), which activates caspase-8, which triggers effector caspases (the executioner) to cleave their substrate protein and drive the cell into apoptosis
Intrinsic pathwaY: internal signal activates mitochondria to release cytochrome C. Cytochrome C and Apaf-1 (apoptotic protease activating factor) bind together, and with the help of ATP generates the apoptosome, which activates caspase 9, which in turn activates the effector caspases (like caspase 3)
Caspases
Initiators: 2, 8, 9, 10
executioners: 3, 6, 7
Activation of procaspases
Inactive procaspase is activated by dimerization and auto-proteolytic cleavage
DNA replication
cellular DNA must be copied before a cell can divide
each strand serves as a template for a complementary strant (AT + GC). Semiconservative (old strand + new strand = new double helix)
DNA is synthesized in 5’ -> 3’ direction
replication is discontinous on the lagging strand (Okazaki)
Replication is initiated by the DNA polymerase alpha-complex, that generates a 7-10nt RNA primer (primase) extended by POLalpha
POL epsilon synthesizes the leading strand, POL delta the lagging
The helicase separates the strands, replication protein A (RPA) binds ssDNA and prevents reannealing
Supercoiling in front of the replication fork is twisted up by the topoisomerase, which releases topological stress
PCNA
Proliferating cell nuclear antigen
DNA clamp essential for DNA replication and coordination of the relication machinery
trimer that encircles the newly replicated DNA (dsDNA)
binds/coordinates the polymerases and other factors
important for processivity
master coordinator (hub) that can bind several hundred proteins involved in DNA replication, DNA repair, DNA damage tolerance (TLS), chromatin remodeling, cell signaling
proteins that binds pCNA contain PCNA-interacting motifs (PIP, APIM)
interaction is regulated by PTMs on PCNA and/or target proteins
Might be target for anti-cancer therapy??
CMG helicase - 11 subunit molecular machine
C = CDC45, M= MCM2-7 ring, G=GINS (heterotetramer)
MCM2-7 ring (heterohexamer) “ATPase motor, encircles ssDNA (template for leading strand) and travels 3’->5’ direction
CDC45 and GINS (heterotetramer) assessor factors are important for posiitoning
Topoisomerase - the topological stress releaser
Topoisomerase inhibitors are widely used in cancer treatment, often in combination with other cytostatic drugs
Irinotecan: colon cancer (+5 FU), small cell lung cancer (+cisplatin)
Etoposide: testicular cancer, lung cancer, lymphoma, leukemia, neuroblastoma, and ovarian cancer
Doxorubicin: breast cancer, bladder cancer, lymphoma, and acute lymphocytic leukemia
What happens at the chromosome ends (telomers)?
no obvious way to copy the most terminal sequences of the 3’ end, RNA-primer cannot be formed (boyond the end)
the chromosomes become slightly shorter for every round of replication/cell division
related to natural “ageing”
Embryonic stem cells, sperm cells, actiavted lymphoytes and many cancer cells express telomerase
telomerase is a cellular reverse transcriptase that extends the ends of linear chromosomes (telomeres). It contains an RNA component to which the ss 3’end of the chromosome hybridizes. Telomerase then adds several nucleotides
Most somatic cells do not express telomerase, and therefore tolerate ca 50-70 cell divisions
Telomerase mechanism
telomerase binds to 3’ flanking end of telomere that is complementay to the telomerase RNA
bases are added putside the strand using RNA as template (telomerase only binds part of its telomer-RNA to the DNA; the rest is left “open” for bases to be added on
telomerase relocates to the new end of the strand
second step is repeated
DNA polymerase complements the lagging strand
Gene expression
A multistep process in which the genetic informaiton is used to produce a functional product - RNA or protein
Gene expression and its regulation are the fundament for cellular and organismal life - gene expression defines the cell type and function
Two main processes - trc and trn
Transcription
The process where genes in the genome are copied into ssRNA molecules by RNA polymerase
transcription
initiated at promoter regions - binding sites for general TFs
regulated by TFs (ca 10% of coding genes, DNA binding) and chromatin structure (epigenetic regulation)
Most RNA molecules are modified after transcription
Maturation of pre-mRNA to mRNA:
- 7mG-Cap
- polyA-tail
- removal of introns by splicing
tRNAs
delivers AAs to the ribosome
transcribed (POLIII) as primary transcripts (1-5 tRNAs)
processed by RNase P (ribozyme) and endonucleases (RNqse D)
primary transcripts may contain introns adjacent to their anticodons that are removed
tRNA nucleotidyl-transferase adds CCA at the 3’ end
high content of covalent modifications (25% of the nucleotides)
protein degradation
eliminates abnormal proteins
- lysosomal degradation (organelle, acidic pH, more than 50 hydrolytic enzymes (proteases), non-selective)
- targeted degradation of proteins marked with ubiquitin by the proteasome (ATP-dependent)
proteasome inhibitors: drugs that block the action of proteasomes. Approved for use in treating multiple myeloma
methods in molecular medicine
separation (reduce complexity of a biological sample), electrophoresis (1D or 2D), chromatographic steps
isolation/purification (DNA; RNA; proteins): extraction, precipitation, chromatography
detection of specific DNAs, RNAs or proteins: Southern blot (DNA), Northern blot (RNA), Western blot (protein), MS
amplification:
- DNA/RNA - PCR and/or cloning
- proteins - cloning followed by heterologous expressionn (bacteria, yeast, insect cells)
sequencing (DNA/RNA)
genome engineering (CRISPR/Cas9)
cellular localization and trafficking (confocal mmicroscopy)
NTNU covid test
- mucus sample from nose/throat
- sample is mixed with a chemical solution that causes the viral protein envelope to crack open
- magnetic nano-particles are added. virus RNA binds to the particles
- magnet pulls the RNA and nano-particles from the solution
- the RNA is released from the nanoparticles for analysis
- the RNA is analyzed in a PCR machine
CRISPR/Cas9
clustered regulatory interspaced short palindromic repeats
RNA-guided nuclease (Cas9) that generates a ds break at the site that matches the RNA sequence
break is repaired by non-homologous end-joining (NHEJ), which is error prone
the “repair” results in indels, deletions, out of frame, premature stop codon, no active protein product
