Unit 2 - DNA/Protein synthesis/ Cell signalling/ Cell Proliferation Flashcards
What is the structure of DNA?
-DNA nucleotides (phosphate, sugar, nitrogenous base)
-Double helix, 2 antiparallel strands 5’ to 3’
-Held together by complimentary base pairs via hydrogen bonds
What is a gene? What is it made from?
-Section of dna that codes for a sequence of amino acids to make a protein.
>Introns not expressed : Exons expressed
What is the organisation of genetic material?
1.DNA tightly packed, well organised and accessible
- coiled many times around histone proteins (structural support) - nucleosome
>DNA is packed in chromosomes
What are the 3 types of chromosomes?
- Metacentric
- Submetacentric
- Acrocentric
What is a karyotype? What can you do with them?
-A karyotype is the 22 pairs of autosomal chromosomes and the 2 sex chromosomes
1. Chromosomal aberrations
- Extra or fewer chromosomes
- Duplications
- Deletions
- Translocations, etc.
- Taxonomic relationships
- Information about evolutionary events
Describe the process of semi-conservative DNA replication. INITIATION
INITIATION:
1. Topoisomerases unwind DNA supercoil
2. Helicase separate DNA strand by breaking h-bonds
(SSB’s coat single strand DNA to prevent re-annealing)
Describe the process of semi-conservative DNA replication. ELONGATION
> Leading strand : DNA primer binds so DNA polymerase can add nucleotides
- Synthesises short amount of DNA so uses sliding clamp to keep it bound to DNA (PCNA) - continuous strand
Lagging strand: DNA polymerase dissociates itself from the clamp and re-associates with new clamp assembled by DNA primer on the next fragment (fragments joined by DNA ligase)
What about the ends of the chromosome?
- Telomeres at the end of chromosomes with no informational role
> Recognised by SSB’s that attract telomorases which recognise tip of existing telomere repeat and elongate protecting end of chromosomes
What are the 3 ways that fidelity of DNA replication is maintained?
- Structural differences of purines/pyrimidines : Correct nucleotide has a higher affinity. DNA polymerase active site only accommodates properly matched base pairs.
- Proof reading activity of DNA polymerase :
-DNA polymerase has 3’-5’ exonuclease activity
> checks last nucleotide added - Mismatch repair :
-Recognise mismatch and correct in new daughter strand
-DNA polymerase clips the incorrect nucleotide off and replaces with the correct
What happens during transcription?
- TF2D binds to promotor sequence : TATA box
> causes distortion of the DNA at TATA box which
causes of a physical landmark of the promoter
» Transcription factors position RNA polymerase at the promoter sequence ( TATA box) - Transcription initiation complex (TF+ RPII)
- TF2H parts double helix at the start point exposing template strand , also enables RPII to change conformation allowing it to be released from the complex and move downstream starting elongation process
What are enhancers and mediators ?
- High level of transcription = more binding of transcription factors on additional sites > enhancers
(increase level of transcription) - Mediators > physically link TF and other components of initiation complex to RPII
What are the modifications of pre-mRNA?
>Describe these
- 5’ end is modified to form a cap
- 3’ end is adenylate
- RNA splicing removing introns
-All introns begin with GU and end with AG
-2 cuts occur
1.splice donor site at the 5’ end of the intron
2.splice acceptor site at the 3’ end of the intron
- Via Spliceosomes (snRNAs)
How is mRNA exported from the nucleus?
-Proteins signify maturity of mRNA
-Exported from nucleus into cytosol for translation via Nuclear pore complex
- with specific nuclear transport receptors through channels in the nuclear membrane
-Then it dissociates when through pore
What happens in translation?
-Ribsome has 3 sites for tRNA to bind and 1 for mRNA to bind (APE)
-No tRNA carries anticodon complimentary to stop codons, ribosome is dissembled and polypeptide is released
*Ribsome travels along mRNA sequence
What is cell signalling? How does it relate to disease?
- Process that allows cells to maintain homeostasis
> defects in cell signalling can cause disease e.g. by mutations, pathogenic organisms
Describe the process of cell signalling resulting in a cellular response.
1.Release of a ligand (by a signal sending cell)
> Epidermal growth factor (EGF)
2. Ligand delivery and binding to the specific receptor (of signal receiving cell)
> EGF binds EGF receptor (EGFR)
3. Receptor activation and signal relay inside the cell
> EGFR activation and relay of signal through phosphorylation
4. Further relay of signal by intracellular signal transduction proteins
> protein-protein interactions
5. Activation of target protein and cellular response
> Transcription factor activation and gene expression,
> cellular function
6. Termination of signal
> Decrease levels of ligands/receptors, dephosphorylation
What is
-Paracrine signalling
-Endocrine signalling
-Contact dependent signalling
-Autocrine signalling
-Paracrine signalling : Nearby cells
(Nerve cells signalling : send signals further away via long axons = synaptic signalling)
-Endocrine signalling : Via circulatory system distributed to any part of the body
-Contact dependent signalling : Close proximity , signalling protein acts as a ligand while still bound to membrane and directly binds with receptor on adjacent cell.
-Autocrine signalling : Cell produces signalling molecule but also has corresponding receptor too, self dependent
How do enzyme- coupled receptors work?
How do G-protein coupled receptors work?
What are enzymes, protein kinases , on and off signalling proteins and phosphorylation?
- Enzyme: Biological catalyst - Control speed of cellular response, faster mechanisms involve already synthesised enzymes e.g. activation via phosphorylation
- Kinases: Phosphorylate other proteins - differ depending on the AA they add the phosphate group
- OFF = G-proteins inactive when GDP bound
ON = removal of GDP and binding of GTP activates G-proteins
(GEF - guanine exchange factor allows GDP to replace GTP)
(GAPS - GTPase activating proteins increase GTP hydrolysis and activates G-proteins ) - Phosphorylation is the addition of a phosphate group from ATP to amino acids by protein kinases.
Phosphorylation can either activate or inactivate a protein.
Dephosphorylation by phosphatases removes it
What are the 3 types of GPCR?
What happens when a ligand binds to GPCR?
- Allows it to bind to G-proteins to further relay the signal.
-G-proteins provide a regulatory point in GPCR signalling since different G-proteins function differently causing different downstream signalling pathways to be activated/deactivated.
What are the 3 subunits of G-proteins and how does it determine their function?
How does GPCR signalling increase blood glucose level?
1.In a resting cell the Gs-protein is inactive. Its α subunit is bound to GDP.
2. When signalled by a ligand, GPCR becomes activated and this allows it to bind to the G-protein via the α subunit.
3.GPCR activates the α subunit of the G-protein and this involves displacing GDP for GTP.
4.Active α subunit disassociates from the βγ pair and binds to adenylyl cyclase to activate it.
5.AC is also an enzyme, it makes cAMP using ATP. Once activated, AC is able to increase cAMP concentration by 20 fold in seconds
6. cAMP is a second messenger. cAMP is used to activate Protein Kinase A. PKA is also an enzyme ( and a kinase) and phosphorylates other proteins e.g. glycogen phosphorylase.
7.Glycogen phosphorylase breaks down glycogen to produce glucose.
How does cell signalling work via Gq proteins?
-IP3 + DAG second messangers
What is cell proliferation? Why is important we control it?
-Increase in cell number
-Deregulation:
Cancer : hyperproliferation
Neurodegeneration: loss of neuronal cells via apoptosis
How are cells signalled to divide?
-Mitogens
-Cause synthesis of proteins to overcome brake so cellular division can pass the R-point
What are the 3 components of MAPK?
- 3 proteins all kinases , known by the final kinase e.g. ERK
-MAPK cascades relay a wide range of signals resulting in different cellular responses.
How does a mitogen outside the cell tell it to divide?
- EGF is a mitogen which signals cells to enter the cell cycle. EGF binds to EGFR to phosphorylate
it and cause signal relay. - Signal is further relayed through the ERK-MAPK signalling pathway.
{Grb2 (adapter protein) + SOS (GEF) activate RAS - RAS (g-protein) activates MAPK signalling cascade} This leads to the activation of AP-1. - AP-1 causes the expression of proteins which cells require to pass the R-point, enter the cell cycle and proliferate.
How is mitogenic signalling turned off?
1.Turn off signalling source: Prevent release of ligand (EGF). EGF is normally synthesised as a transmembrane protein. During a cellular response like proliferation metalloproteinases become activated which cleave and make EGF diffusible.
2. Turn off receptor : Endocytosis > EGFR ubiquitinated marked for degradation > delivered to endosome which fuses with lysosome and deregulation of receptor occurs.
Cbl* functions in the endocytosis of signalling receptors and is thought to be an important protein to prevent the formation of cancer. Explain how mutation of the cbl gene could contribute to the development of cancer.
EGF
EGFR
1.Endocytosis is required to turn off (down-regulate) signalling receptors like EGFR.
2.Mutation of Cbl would cause defect in the endocytosis process and block the downregulation of EGFR.
3.Block in endocytosis of EGFR would allow it to remain active.
4.This would mean that EGFR would be able to signal continuously enabling cells to divide /enter the cell cycle/proliferate and lead to development of cancer.
B-Raf is a subtype of the Raf protein and relays signal to MEK. B-Raf undergoes a driver mutation which allows it to be activated without receiving a signal from Ras. The mutation increases its activity 500 fold and this is found to be a common mutation in some cancers.
State what type of mutation B-Raf undergoes and explain how this could contribute to cancer development.
1.B-Raf undergoes gain of function mutation.
2.The B-Raf mutation allows it to be active without the cells receiving any mitogenic signal.
3.This would result in continuous signal being relayed to ERK and activation of transcription factors
4.causing continuous expression of proteins involved in activating the cell cycle, resulting in hyperproliferation and the development of cancer.
How can you treat B-raf mutations?
- Raf inhibitors block its kinase
activity - This blocks MEK and ERK activation
- Resulting in a block in the cell cycle
This causes cells to die as a result of apoptosis being triggered
What is apoptosis? What does p53 do?
-Eliminates cells that have become abnormal or damaged caused by DNA damage.
> TF - prevents cells with damaged DNA to proliferate further
How are cells kept under control and apoptosis not triggered by mistake?
- p53 levels kept low , continuously synthesised and destroyed
-After synthesis it is bound by protein Mdm2
> Tag for degradation with ubiquitin differently to receptors which ensures it is delivered to proteasome for degredation
How des p53 act as a tumour suppressor gene?
What is the role of p38 MAPK signalling pathway?
-The p38 MAPK can be activated by cellular stress (including DNA damage) AND can mediate apoptosis too.
-p38 acts as a tumour suppressor. Loss of p38 function may play an important role in cancer development.
What is the JNK MAPK signalling pathway?
What role do cell cycle regulators play in DNA replication?
- Activation of cdk1
- Active Cdk act on different proteins to trigger events such as cell cycle entry, DNA replication, spindle assembly.
-Active > Bind to cyclins = cdk can now phosphorylate substrates
How does cyclin and cdk act as a series of biochemical switches?
The R-point is controlled by what protein?
-Rb protein (retinoblastoma)
- To advance from R-point Cyclins are needed which are made from active E2F transcription factors.
-R- point E2F are inactive as they are locked onto Rb , preventing E2Fs to express the required cyclins for cell development
Describe the process of how do cells advance from G1 phase?
-Hyper-phophorylate Rb to inactivate
1. Mitogen signalling activate Transcription factors such as AP-1
2. AP-1 binds to DNA and codes for cyclin D mRNA
3. Cyclin D mRNA is translated to produce cyclin D
4. Cyclin D binds and activates cdk 4/6
5. Cyclin D cdk 4/6 phosphorylates Rb which INACTIVATES Rb releasing inhibition on E2F
6. E2F is now active and induces gene expression of cyclin E and others….
What are Cdk inhibitors and their functions? Specifically highlighting 2.
-Cdk inhibitors bind to cyclin-Cdk complexes and block their action and hence block cell cycle progression.
1. p16INK4 : Target = Cyclin D-Cdk4/6
Activated in response to growth inhibitory signals
Function: prevent passage of R-point
2. p21CIP : Target = Cyclin E-Cdk2 / Cyclin B-Cdk1
Activated in response to DNA damage
Function: CELL CYCLE ARREST
Describe normal process of cell proliferation?
- Mitogen > Signalling cascase > Cyclin D + E > Hyper-phosphorylation of Rb > inactivates Rb > Cell G1 to S phase
- Cyclin A-Cdk2 triggers events resulting in DNA replication. > DNA replication + Protein synthesis of chromatin to package DNA into chromosomes
- G2 > Cyclin B-Cdk1 is formed during G2 phase and allows cells to progress to M phase > delay for growth and check DNA is faultless
- M-phase > Cyclin B-Cdk1 phosphorylates proteins which result in
- condensing of DNA
- formation of the mitotic spindle
- breakdown of the nuclear envelope.
> Preparation for chromosome segregation, correct attachment of sister chromatids to the spindle. - Spindle assembly checkpoint
Unattached kinetochores to the spindle is detected by the SAC which blocks entry to anaphase. - Cyclin B-Cdk1 and APC in the separation of chromatids > Cyclin B-Cdk1 activates anaphase promoting complex (APC) which destroys cohesions holding sister chromatids together
- Deactivation of cyclin B-Cdk1 by APC results in spindle disassembly and reformation of nuclear envelope. The cytoplasm is divided into two cells.
Upon mitogenic signalling, for each of the four phases of the cell cycle name a main step or a control point and the relevant cell cycle regulator/protein.
- G1: Pass R-point
- mediated by cyclin D-cdk 4/6 - S-phase : Replicate DNA
-mediated by cyclin A-cdk 2 - G2 : G2/M control point
- to pass requires action of cyclin B-cdk1 - M-phase : Mitotic spindles form by action of cyclin B-cdk1
The retinoblastoma gene (Rb) is found to be mutated in many types of human cancer. What effect does Rb mutation have on the cell cycle
- Rb genes codes for Rb protein which is a tumour suppressor gene - controls the R-point
- Rb active it Inihibts E2F - Prevents the expression of cell cycle regulators e.g. Cyclins
- Mutation of Rb inactivates it - cell can enter cell cycle without mitogenic signalling
How does the cell respond if DNA is damaged?
-Kinases acting as sensors detect DNA damage - phosphorylate P53
-P53 is a tumour suppressor gene acting as a transcription factor
1. P53 causes expression of cdk inhibitor p21 - halts cell cycle
2. Coordinates expression of DNA repair enzymes/ proteins - repair damaged DNA
3. Express apoptotic proteins - trigger activation of apoptosis
What is cell apoptosis? What do caspases do?
-Programmed cell death > Avoids inflammatory response
-Caspases cause series of distinct cellular changes:
1. Cleave proteins of the cytoskeleton, this results in cells to undergo morphological changes and blebbing of the membrane (membrane bulging out).
2. Cleave proteins of the nuclear envelope (membrane) causing the nucleus to breakdown.
3. Caspases activate nucleases which cut DNA into fragments (DNA fragmentation).
> The cell eventually breaks up into several apoptotic bodies.
The apoptotic bodies end up being cleared by macrophages through phagocytosis.
How is apoptosis activated by DNA damage?
