Random Flashcards
Describe the action of the nitrogenase enzyme
Fe protein accepts electrons from ferredoxin and can act as a reductase.
ATP hydrolysis makes confirmational change in that brings the two proteins closer together (2Fe-S clusters and 2Mo-Fe clusters used to reduce N2 to NH3)
How is oxygen reduced around nitrogen fixing bacteria in root nodules?
Leghaemoglobin: has high affinity with oxygen.
How is ammonia aquired in plants without nitrogen fixing bacteria?
Reduction reactions
Nitrate -> Nitrite -> Ammonia
Electrons for reduction come from NADH or NADPH
Enzymes: Nitrate reducase and Nitrite reductase
Both enzymes make use of an Mo cofactor
What is the final product of amino acid biosynthesis from ammonia?
Glutamate - but there is two routes to get to this
2 methods for producing glutamate from ammonia
Animals and fungi: Use glutamate hydrogenase to reduce alpha-ketogluterate using NADH or NADPH
Plants: Synthesise glutamate from itself. Glutamine synthetase enzyme used. Used NADH reducion power.
Glutamate -> Glutamine
Glutamine + Oxoglutarate -> 2 glutamate
2 ways to omake amino acids from glutamate
- Transamination (transfer of amino group between two keto-acids) amino transferases
- Carbon skeleton alteration
Where do carbon skeletons for amino acid synthesis come from?
The TCA cycle: specifically oxoglutarate
How are the carbon skeletons being removed from the TCA cycle replaced?
Carboxylation of C3 intermediates of glycolysis
Pyruvate + HCO3 -> Oxaloacetate
Pyruvate carboxylase
Therefore carbon enters the TCA cycle as OAA or acetyl coA
Describe the action of lysozyme
Cuts the polysaccharide chain in peptidoglycan of bacterial cell walls
Hydrophobic active site is large enough to fit 6 sugar residues. Ring D becomes strained when bound to the enzyme and forced into a half chair structure.
Wall is cut between D and E rings (nucelophillic attack)
Inhibitor of F-type ATPase?
Oligomycin
Evidence for chemiosmosis?
Uncoupling proteins
Inhibiting ATPase with oligomycin stops the electron transport chain.
Detergents stop proton pumping and ATP is not produced.
ATP produced using an artifically generated proton gradient.
What is a haplotype?
A set of SNPs inherited together: can be informative for human disease.
How was the gene causing cystic fibrosis identified? (a monogenic disorder)
- Identifcation of an RFLP that segregated with the disease
- Identify the chromosome: long arm of chromosome 7 (done by fusing with mouse cell - as the mouse cell divides, it looses human chromosomes)
- Chromosome walk narrowed down the region of interest to 500kb
- Used a mixture of northern blots and zoo plots to identify the gene: a gene that codes for a transmembrane chloride channel
66% of CF occurences are due to one mutation: missing phenylalanine.
1525 different mutations in the gene have been idenifed, making diagnostic screening difficult.
How does Dictyostelium aggregate?
Founder cell emits pulses of cAMP
cAMP binds to GCPR activating AC and PLC
cAMP: causes more cAMP to be release
IP3: releases calcium to catalyse chemotaxis (cytoskeletal rearrangement)
Define a secondary messanger
A small molecule that is formed in or released into the cytosol in response to an extracellular signal and helps to relay the signal to the interior of the cell
Describe Tyrosine Kinase Linked Receptors with example
Example: platlet derived growth factor
Binding of PDGF induces dimerisation of 2 receptors
Tyrosine residues phosphoylate each other and these activated residues activate
- PLC
- GAP
- SOS
How does Ras control gene expression?
- A monomeric G protein
- Activated by GTP binding (catalysed by SOS enzyme)
- Initates a phosphoylation cascade that results in MAP kinase activiation
- MAP kinase is able to regulate transcription of proteins involved in the cell cycle (cyclins etc.)
Examples of communication in unicellular organisms
cAMP acting as aggregation stimulus is Dictostelium
Quorum sensing in bacteria (Lux proteins regulate light emission and light is only emitted when a critical population density is reached)
Quroum sensing
Light only expressed at high population densities in Vibro fischeri
LuxI operon expressed during exponecial growth and causes systhesis of OHHL
OHHL can therefore be used as a signal for the popuation density of the bacteria
OHHL concentration sensed by LuxR
At critcal density, LuxR induces LuxL (more OHHL synthesis - positive feedback) and LuxAB which causes light to be emitted.
Protein pairs used in environmental sensing in bacteria
Sensor and response regulator
Sensor: is phosphoylated by input signal
Phosphate is physically transfered to the response regulator
What are the 4 sets of proteins used for chemotaxis in e.coli?
MCPs: Membrane bound signal transducers
Cytoplasmic signal transducers: CheA, CheY, CheW and CheZ
Flagellar switch
Adaptors: CheR and CheB
Adaptation of chemotaxis in bacteria
Results in “random biased walk”
CheR = methylator of the receptor
- methylated receptor is less active leading to cheA to return to phosphoylated state (after dephosphoylation was caused by attractant binding)
CheB = demethylator
- activated by CheA-P (which phosphoylates CheB)
- in the presence of a repellent
Plant communication examples
- ABA causing stomatal closure
- Giberellins and growth gene expression
- Phytochromes
How does ABA cause stomata closure?
ABA inhibits PP2C
PP2C can no longer inhibit OST1
OST1 activates Cl efflux and inhibits K influx
What is photomorphogenesis?
Light dependent determination of plant growth
- Impacted by Phytochromes (increase in the nucleus in the light)
- COP1 increases in the nucleus in the dark
Photomorphogenesis is repressed in the dark.
How does phytochrome impact photomorphogenesis?
Active phytochrome enters the nucleus and phosphoylates PIF3
PIF3 is degraded when phosphoylated
PIF3 usually downregulated photomorphogeneis
Overall: Active phytochrome stimulates photomorphogenesis
How does COP1 negativly regulate photomorphogenesis?
COP1 is an E3 ubiuitin ligase
Targets HY5 for degregation
HY5 activates photomorphogeneis
COP1 accumulates in the dark and is exported from the nucleus in the light.
How does GA target DELLA for degregation?
GA binds to GID1 which recruits E3 ubiquin ligases to target DELLA for degregation
Example of convergence in plant signalling
GA and Phytochrome signalling converge on PIFs to regulate growth
- GA accumulates in the dark
- Phytochrome is active in the light
Action of PIF3 and HY5
PIF3
Inhibits expression of photomorphogeneis genes
Increases expression of growth genes
HY5
Activates photomorphogeneis genes (broken down by COP1)
Examples of communication in animals
Adrenline (cAMP signalling with Gs protein)
Control of blood pressure with NO
Egg fertilisation
Mechanism of heterotrimeric G protein activation
Binding of stimulus to GCPR triggers confirmational change in the G protein that favours the binding of GTP instead of GDP to the alpha subunit
GTP binding causes alpha subunit disassociation
Alpha subunit binds to adnylate cyclase and activates it
Heterotrimeric G proteins have ATPase activity and so GTP is hydrolysed and the alpha subunit reassociates with the beta and gamma subunits
How does caffine affect cAMP?
Inhibits phosphodiesterase which usually breaks down ATP
How does cholera toxin affect cAMP?
GTPase activity of G protein inactivated
Therefore Ga remains bound to adrenlylate cyclase and there is no inactivation. Lots of cAMP causes flow of water into the small intestine resulting in diorrhea
How is NO synthesised?
- Stretch activated calcium channels open when blood pressure is high and calcium activates NO synthase
- ACh binds to GCPR, activating PLC and IP3 causes release of calcium from the ER to activate NO synthase
Effect of NO on smooth muscle
Activates guanylyl cyclase
GTP -> cGMP which causes relaxation
Egg fertilisation (describe)
- Acrosome reaction (sperm causing the breakdown of the zona pullucida)
- ZP3 receptors reconised by the head of the sperm: leads to increase in Calcium
- Calcium increase releases hydrolytic enzymes to degrade the zona pellucida - Egg activation (stopping polyspermy)
- Phosphoinistol pathway used to release IP3 and calcium released
- Burst of calcium triggers coritcal granules to be released to block another fertilisation event
- Activation of first cell cycle proteins (cyclins and cdc2)
Why is calcium a good signalling molecules?
Concentrations in cytosol are kept low as calcium is stored in the ER
Makes it an energetically cheap second messenger
Very few ions need to move into the cytosol to generate a reaction
Example of notch-delta signalling
If a neural cell begins to develop in drosophilia it presents a *delta ligand** on it membrane.
Notch receptors on adjecent cells bind to the delta ligand and development into a neural cell will be prevented
What factors affect the phase transition temperature range of a membrane?
Carbon chain length: shorter carbon chains have a lower melting point
Fatty acid saturation: Unsaturated fatty acids have kinks which lower the melting point (weaker van der waals forces)
Sterols: Prevent chains from packing too closely below transition temperature
Restricts head group movement above transition temperature
How does SDS polyacrylamide gel electrophorisis work?
SDS = anionic detergent
Seperates polypeptides
Denature the proteins by adding reducing agent
SDS binds to amino acid chains and makes them negativly charged
Run on gel to seperate proteins by weight
Experimental evidence for flip flopping of lipids
Spin-Label the whole thing
+ Acsorbate which removes the label from outer lipids (signal will fall by half)
Wait
+ Ascorbate and the signal will fall even furthur (unlabelled phospholipids have moved to the outside)
How to test for protein mobility in a membrane
Cell fusion experiments: label mouse and human antibodies with different colours and fuse cells. See how label distrubution changes
FRAP: floresecnce recovery after photobleaching.
- Flourecence over whole cell
- Bleach half the cell
- See if flourence is recovered
How to find out if a protein spans the membrane?
Sample 1: Radio label proteins on the outersurface
Sample 2: Make membrane permeable to the label radio-label (inside of the protein would also be labelled)
Extract the protein and detemrine if sample 2 has more radiolabels than sample 1 (SDS polyacrymide gel)
How is protein fate determined (proteins that are not meant to stay in the cytoplasm)
Signal sequence: 15-30 amino acids long at the N-terminus to direct the protein into the ER.
- Signal sequence reconised by signal recognition particle when the protein is being made by the ribosome
- SRP binds to the ER at SRP receptor
- SRP dettaches
- Protein synthesis is resumed by the ribosome
- Protein is released into the lumen of the ER
**intergeral proteins contain “stop-transfer sequences” that stop translocation so they remain in the membrane
How are proteins directed to final position in the cell
Retention sequences: stay in ER
Sorting sequences: move to different cell comparetments
No sequence: Exocytosis
Why is transport across a cell membrane important?
Communication
Volume regulation
Compartmentalisation
Describe receptor mediated endocytosis
Binding of receptor stimulates adaptin protein to bind and clatherin to accumulate beneath adaptin.
Clatherin polymerises to form coated membrane pits
Confirmational change in receptor releases cargo into the clatherin coated vesicle
When does the transport of substances across a membrane happen spontanously?
Negative delta G - the process is exergonic
[X]i < [X]o
delta G = RTln([X]i / [X]o)
How is the rate of diffusion determined for small uncharged molecules?
Permeability coefficent (how permeable it is in the membrane) and the concentration gradient.
Types of ATPase
P-type (sodium potassium pump)
V-type (found in endomembrane and pump protons - eg. vesicle membranees)
F-type: F0 and F1 domain
Explain how the lac operon is regulated.
Low glucose: Increased cAMP causes increased binding of CAP. CAP bound to the operon increases the effectivness of RNA polymerase binding.
High lactose: Lac operon repressor is inactivated by lactose.
Repressor: Lacl (inactivated by lactose)
Activator: CAP (activated by cAMP when glucose is low)
Describe translation initation
Prokaryotes
- Shine delgardo binds to 16S subunit rRNA
- 30S subunit recruited
- IF2 + GTP hyrolysis recruits the 50S subunit
Eukaryotes
- eIF3 + 40S + tRNAmet + eIF2 = 43S complex that binds to 5’ cap
- 60S subunit recruited by GTP hydrolysis
Describe translation elongation
EF-Tu:GTP:aminoacyltRNA enters the A site of the ribsome
GTP hydrolysis takes place if the codon and anticodon are complimentry
Peptide bond formed
EF-G:GTP is recruited and GTP hydrolysis aid translocation of the ribosome
Cost: 1 ATP (to charge the tRNA)
2 GTP
GTP is bound to EF-Tu and EF-G
Describe translation termination
Releasing factors recognise stop codons
RF3 binds to GTP
GTP hydrolysis removes the ribosome
Examples that show linkage
Bateson, punnet and saunders: Pollen shape and flower colour
THM: purple eyes and vestigal wings in drosophilia
How can virus’s interfer with the host cell cycle?
Reduce regulation of the cell cycle by interfering with cyclins, CKIs etc
Stop host cell apoptosis from taking place
HPV: produces E6 and E7 oncoproteins
E7 upregulates G1-S cycles
How does the RB protein effect the cell cycle?
Contols the restriction point
A negative regulator of the cell cycle.
Regulates the movement into S phase
Binds to E2F, which prevents movement into S phase.
Is phosphoylated by G1-S cyclins/cdks which causes it to be removed and S phase can progress.
If there is a mutation, and it is inactivated, the cell cycle can progress through G1 and S without regulation.
Difference between negative regulators and checkpoints in controlling the cell cycle?
Negative regulators
eg. RB (retinoblastoma) - loss will deregulate the restriction point
checkpoints/apoptosis
eg. p53 DNA damage checkpoint (activates pip21/cip1 which inhibits cdks for progression to S phase) and promotes apoptosis (bax protein)
Why is glycolysis exogernic?
Produces ATP
ex: phospholenolpyruvate has a lower gibbs free energy than ADP, so it is able to phosphoylate ADP to form ATP
How are reactions made more thermodynamically favourable?
Activated carriers
ATP
Acetyl-coA
NADH
FADH2
Advantages and disadvantages of futile cycles?
Good: can rapidly increase flux through a pathway. Increase in Fructose-2,6-bisphosphate increases rate of glycolsis
Need to be tightly regulated to prevent energy being wasted
Define a centimorgan
The distance between two genes at which recominant frequency is 1%
Morgans 3-factor mapping experiment
Make F1 generation (AABB x aabb)
Backcross with recessive parent (AaBb x aabb)
Score phenotypes (due to crossing with resessive, phenotypes will directly correlate to genotypes).
Generally explain how a eukaryotic transcription factor may work
May bind to DNA and dimerisase. This binding can be used to help recruit general transcription factors.
Can also recruit histone acetyl transferases, which acetylate nearby histones to open up the chromatin structure.
What cleaves RNA strand from okazaki fragments in eukaryotic DNA replication
FEN-1
What are the proteins that recognise site for splicing?
U1 and U2 snRNPs
Recruit the sliceosome
Difference between standard gibbs free energy change and that seen in a cell
deltaG = deltaG’ + RTlnk
How to tell if mutations are in the same gene or different genes?
Do they compliment each other?
Yes - different genes
example: Benzor T4 bacteriophage and the rII gene. Two different mutants cannot kill e.coli K alone but can if recombination takes place and the genes can compliment each other
Where are restiriction enzymes produced?
Produced by bacteria - to break down incoming phage particles
Markers of a good plasmid vector
Site of replication
Selectable marker
How to construct a genetic library?
Partially digest genomic DNA
Insert into phage lamda with BamHI restriction enzyme
