QBIO2001 Flashcards
Modelling
1
Q
Why are there different kinds of cells?
A
Because of their unique proteins
2
Q
How many cells are there in the human body and how are they made?
A
37 trillions made by 10 billion cells/hr
3
Q
What is DNA and what is it made of and what is its order?
A
• DNA (Deoxyribose nucleic acid)-a long, thin thread-like macromolecule which is the information carrying part of the chromosome
• A DNA molecule is shaped like a double helix and is made of two strands (held together by weak hydrogen bonds in the centre) of monomers called nucleotides
• Each nucleotide consists of 3 parts:
o A phosphate
o A sugar
o A nitrogenous base
• There are four types of purine bases:
o Adenine
o Thymine
o Guanine
o Cytosine
• These bases are arranged in a sequence along each DNA strand in a particular manner: adenine always pairs with thymine and guanine always pairs with cytosine.
• The vertical sides of the DNA molecule are made of alternating sugar and phosphate molecules
• The strands go from 5’ to 3’ (where ‘=prime).
• The two strands are arranged in an anti-parallel fashion (with the 5’ end of one strand at the same end of the double helix at the 3’ end of the other strand)
4
Q
How does DNA replication occur?
A
- DNA gyrase cuts hydrogen bonds between nucleotides
- DNA helicase- makes a replication fork
- Single stranded DNA binding proteins keep the strand from re-annealng
- One DNA strand encodes the leading strand, which forms from its 5’ to 3’ end using DNA polymerase
a. There is continuous replication of the leading strand - Lagging strand forms in pieces called okazaki fragments
a. First, an RNA primase lays down an RNA primer dose to replication fork
b. Then DNA polymerase III lays down new DNA in a 5’ to 3’ direction
c. The process repeats again and again
d. DNA polymerase I replaces RNA with DNA
e. DNA ligase then joins the bits of strand together - Topoisomerase rewinds the DNA
5
Q
Why are proteins folded differently?
A
amino acids can have a negative or positive charge, can be polar or non-polar and can be hydrophilic or hydrophobic
6
Q
How does Ras protein activation work?
A
Inactive Ras-GDP releases GDP via GEF action and allows the binding of GTP to Ras, making active Ras-GTP.
GAP turns the Ras-GTP off by making it release a phosphate group, turning the active Ras-GTP into the inactive Ras-GDP.
7
Q
What ion gives the energy that allows a protein to rearrange its conformation, and why?
A
Phosphate- the p negative charge can give the protein ability to change conformation
8
Q
Kind of activation mechanisms?
A
- Calcium
- Phosphorylation
- Glycosylation
- Nucleotide binding
9
Q
What kind of SNPs are there?
A
o SNPs can be o Non-Coding o Coding Synonymous (silent Non-synonymous (changes amino acid sequence) • Missense (different amino acid) • Nonsense (stop)
10
Q
What are SNPs used for?
A
o Identification and forensics
o Mapping and genome-wide association studies of complex diseases
o Estimating predisposition to disease
o Immigration and citizenship in the UK
o Predict specific genetic traits
o Classifying patients in clinical trials
11
Q
How can SNPs cause cancer?
A
If the Ras is always on even without growth factor, then the cells will always proliferate.
12
Q
What is systems biology?
A
The study of systemic properties in a biological object or process
13
Q
What is modelling?
A
Artificial construct describe by maths that represents processes/phenomena in biology
14
Q
How does hypothesis testing work?
A
- Biological and physiological knowledge and data
- Models of gene regulation, biochemical networks, cells and organs
- Computational ‘dry’ experiments and analysis to screen hypotheses
- Experimental design to test hypotheses
- Development of experimental techniques
- ‘Wet’ experiments to verify or reject hypotheses
- Goes back around
15
Q
What are the 6 types of biological models?
A
- Biological system
- Mental model
- Model scheme
- Process model
- Dynamic model
- Quantitative results
16
Q
What are the 5 steps of modelling?
A
• Goals, scope:
o Scope, objectives
Scope of model includes size (m, cm….) of object looked at (e.g. m for humans, nm for organelles) and time range in which the process is happening (evolution of humans for million years, or high energy transition states for ns)
o Data, prior knowledge
• Model selection:
o Types of model
• Model building or design:
o Variables, interactions
System state: snapshot of the system at a given time
Variable- quantity with a changeable value
o Equations, parameters
Parameters- quantities with a given value
Parameter estimation- obtained from experimental literature or from finding best fit of model to data
• Model analysis and diagnosis:
o Verification
Is the model built right? Is it consistent with the laws of nature?
o Validation
Has the right model been built? Does it fit the data?
o Investigation of behavior
• Model use
o Hypothesis testing, simulation, explanation
o Design, drug optimization
17
Q
What are the 5 mathematical model types?
A
- Static(end point) vs Dynamic(temporal)
- Correlative vs explanatory
- Deterministic (population average) vs Probabalistic (molecular fluctuations)
- Well mixed vs varying through the cell
- Quantitative vs qualitative
18
Q
What are the 6 benefits of models?
A
- Make predictions and extrapolations about experimentally untested cases
- Lead to new hypothesis
- Pull together isolated facts and observations
- Explain non-intuitive system based effects
- Cheap and fast
- A simple model enables insight but a complex model goes into more mechanistic detail
19
Q
What are the formulas for rate of change and accumulation?
A
- Rate of change=flux in- flux out + initial value of x
* Accumulation = total flux in- total flux out
20
Q
What is biochemical kinetics?
A
The study of reaction rates
21
Q
What is mass action kinetics?
A
The rate of a chemical reaction is proportional to the product of the concentrations of the reacting chemical species
22
Q
Why are sugars useful in the body?
A
Can take energy stored in bonds of sugar in order to make ATP
23
Q
Can you make fat from sugar and sugar from fat?
A
o Can make fat from sugar but can’t make sugar from fat
24
Q
What are essential amino acids?
A
The amino acids we need to source externally
25
What is the purpose of metabolism?
* Extract energy from fuels (e.g. sugar)
* Store energy (e.g. fat, glycogen)
* Synthesis (e.g. DNA)
* Eliminating waste material (e.g. urea)
26
What is positive energy balance?
- good situation to store energy --> more energy than you need
27
How do pathways control rate of flux through entire pathway?
o Rate determining/ rate limiting steps
o Regulates the system- feedback and feedforward systems
o Positive energy balance- good situation to store energy more energy than you need
o ATP can increase across threshold value but feedback/feedforward steps regulate this
o System can autoregulate these values, saying “we need more” or “stop we’ve had enough”, stimulating or blocking the pathway
28
When is a reaction spontaneous?
• Gibbs free energy = if <0, more likely to occur (spontaneous reaction)
29
What is Gibbs free energy at equilibrium?
~0
30
What is Gibbs free energy at irreversible steps?
<<0
31
What does ATP do to a reaction?
It increases the chance that a reaction will occur
32
How do you prime for a lower Gibbs free energy?
Add P or CoA
33
Does Gibbs free energy have to have the same along the entire pathway?
No-it can vary
34
What is the irreversible step?
The rate-limiting step; hence it is a good site for regulation
35
Substrates that are more reduced yield ___________ upon ______
Give an example
more energy
oxidation
-Fatty acids store more energy per mole than glucose
36
How is ATP made (in short form) and when is it used?
• ATP is made from breakdown of ‘fuel’: ADP --> ATP
• ATP is consumed by performing work: ATP --> ADP + AMP
o E.g. movement, intracellular transport
37
Comparitively to ADP and AMP, what is the concentration of ATP in most cellls?
• Concentration of ATP in most cells is very high higher than 2 products formed when ATP is used so ATP >>ADP +AMP
38
Do levels of ATP change?
Levels of ATP rarely change- they are kept constant
39
How does the cell regulate ATP levels?
• To regulate level of ATP, calculates ratio of ATP vs ADP/AMP
o Metabolism responds to a change in ratio of ATP vs ADP/AMP
o High ATP demand: ATP consumed, increase in AMP + ADP
o Response: High in ATP generation, low in ATP utilization
o ATP demand regulates metabolism
• Small decreases of ATP could generate big increases of ADP/AMP
40
What does allosteric control mean?
concentration of substrates can drive rate of reaction and products can also control rate of reaction
41
Under what kinetics are enzymes?
Michael-Menten kinetics
42
Where is the Km of most enzymes?
o Most enzymes have a Km near physiological concentration of their substrate- more sensitive to [substrate] at lower [substrate]
43
What is Km?
Km- the concentration of substrate at which the enzyme is operating at 50% of its capacity
44
What happens when there is too much substrate and not enough enzymes, and why?
o Too much substrate and not enough enzymes = plateau
| Enzyme saturated when no more active sites are available
45
At what point do most enzymes operate?
• Most enzymes in cells are operating at a point where there are available active sites to bind so that they can control rate of reaction --> regulates the system
-Not at the plateau part of a graph, but at the linear increase part
46
Where does glycolysis occur?
Cytosol
47
Is oxygen needed for glycolysis?
No
48
Summarise the process of glycolysis
```
Preparation-
• Invests 2 ATP-us es 2 ATPs
• Traps glucose
• Activates glucose
• Cleaves glucose into half
o Ends up with 2 phosphoglyceraldehyde
• Cells can now begin to extract energy out of glucose
• Two molecules of ATP are used to phosphorylate and change glucose in preparation for splitting it into 2 3-carbon molecules (glyeraldehyde-3-phosphate)
```
Payback-
• In the second stage, oxidation of glyceraldehyde-3-phosphate to pyruvate is coupled to ATP synthesis: four ATP molecules are produced, giving a net energy profit of 2 ATP molecules
49
Summarise the glycolysis equation
Glucose + 2NAD+ + 2ATP + 4ADP + 2 phosphate groups --> glycolosis --> 2 Pyruvates + 2NADH + 2ADP + 4ATPs
50
Where does the Krebs cycle occur in the mitochondria?
The matrix
51
Describe the structure of mitochondria and why it is that way
• Outer membrane is porous but inner membrane is impermeable
o This allows intermembrane space (IMS) and cytosol to have similar environment but for IMS and matrix to be different (that is have gradients)
o Function- pumps ions from one side to another generates membrane potential across mitochondria
• Inner membrane:
o Has cristae to increase surface area
o Where oxidative phosphorylation occurs
52
Does the mitochondria need oxygen?
Yes
53
Describe the process of the electron transport chain/oxidative phosphorylation
1. Electrons are passed from NADH to NADH Dehydrogenase. Coupled with this transfer is the pumping of 1 hydrogen ion for each electron
2. Electrons are transferred to ubiquinone (mobile transfer molecule) which moves the electrons to cytochrome b-c
3. Each electron from b-c complex moves to cytochrome c which is a mobile carrier that transfers each electron one at a time, pumping on H+ as each electron is transferred
4. Cytochrome c takes the electrons to cytochrome oxidase, where the electrons, hydrogen and oxygen molecules interact to form water- hydrogen ions are pumped across the membrane
5. Hydrogen pumping creates a gradient, gradient used by ATP synthase to make ATP from ADP and inorganic phosphate
a. ATP is turned by the flow of H+ ions moving down their electrochemical gradient
b. As ATP synthase turns, it catalyzes the addition of a phosphate to ADP, capturing energy
54
Why do we need oxygen in the electron transport chain?
• Need oxygen so that electrons can oxidise the oxygen --> prevents damage from being done to other proteins
55
What is steady state?
• Dx/dt= vin -vout =0
o Vin = vout
• Rate of change is zero
• Amount is steady
56
Using mass action kinetics, what is the rate of a chemical reaction proportional to?
• Rate of a chemical reaction is proportional to the product of the concentrations of the reacting chemical species
57
What is the reaction rate of 0 --> A?
v=k
58
What is the reaction rate of A--> B?
v=kA
59
What is the reaction rate of A+B --> C?
v=kAB
60
What is the reaction rate of 2A --> C
v=kA^2
61
What is the reaction rate of mA+nB -->C?
v=kA^mB^n
62
What do enzymes do?
Increase reaction rate by lowering activation energy
63
What is the total flux of
A+A --> C (reaction rate=k1)
0 -->A (reaction rate=k0)
A -->0 (reaction rate=k2)
dA/dt= k0- 2k1A^2 -k2A
| dC/dt=k1A^2
64
How do enzymes work?
S+E ES -> P+E
65
What is the reaction rate for Substrate --> product reactions using Michaelis Menten?
v= VmaxS/ Km+S
66
What do inhibitors do?
• Inhibitors reduce flux and different types can interfere at various steps
67
How does allosteric regulation work?
Allosteric regulation binds with the enzyme but not at the active site
68
What happens at allosteric activation?
The active site becomes available to the substrates when a regulatory molecule binds to a different site on the enzyme
69
What happens at allosteric deactivation?
The active site becomes unavailable to the substrates when a regulatory molecule binds to a different site on the nzyme
70
What is the reaction rate with allosteric inhibition?
v= VmaxS/ Km+S * 1/1+ I/KI
71
Draw a diagram of a gene
Look at notes diagram
72
Why are genes regulated?
• Genes are regulated so that energy isn’t wasted by the cell by making mRNA for transcription and proteins by translation
73
What do structural genes do?
encodes the mRNA to be transcribed --> these will be expressed as protein
74
What does the promoter/operator genes do?
• Promotor/operator-allows regulatory factors to bind and interact with the DNA
o Promotor- allows mRNA polymerase to bind
o Operator-DNA sequence that the protein products of the regulatory genes can either bind to or not (depends on nutrients) --> binding controls whether mRNA is allowed to be produced and whether the protein is going to be produced
75
What is the regulatory gene?
Constantly produced
o Always expressed
o Act as sensor in order to mediate the regulatory processes
76
Is the operon always on?
No- it can be turned off
77
Why are transcription and translation tightly coupled in bacteria?
Because bacteria don't have membrane separating DNA from ribosomes
78
What is polysome?
a cluster of ribosomes held together by a strand of messenger RNA which each is translating.
79
What are protein properties influenced by?
```
• Physical
o Tertiary structure- Flexibility
Turnover
• Can affect activity
o Secondary structure
Abundance
o Primary sequence
Isoforms
o Mass-pl
Localization
• Chemical
o Solubility- Flexibility-
Cell cycle
o Surface hydrophobicity-
Modofications
o Activity-
Tissue distribution
o Charge distribution
Interactions with other proteins
```
80
Does E.Coli prefer to use glucose first or lactose first? When will it use either and why?
• E.Coli prefers to utilize glucose before lactose
o Will use glucose until it runs out even if lactose is present because the sugar requires no change- don’t have to activate the lac operon it can be used straight away
o When glucose has run out, E.Coli. will sense this, and the lactose present will trigger the lac operon
81
What happens if the lac operon is or isn't activated in E.Coli?
* E. Coli. can’t metabolize lactose in the basal state without activation of the lac operon.
* If the lac operon is activated, it can induce the production of an mRNA which ultimately encodes for multiples proteins, one of which is B galactosidase: this protein can cleave disaccharide
82
What does lacI do?
• Lac I (inducer) – always produced and regulatory gene
| o Binds to operator
83
What does lacZ?
• lacZ-converts lactose into its monosaccharides
84
What does lacY do?
• LacY- allows lactose to get inside E.Coli cell
85
How does the lac operon work?
* In the absence of lactose, Lac I binds to the operator doesn’t allow mRNA polymerase to proceed and produce the mRNA
* When lactose is present, lactose binds to lacI protein that inhibits its ability to bind to the operator region and that can allow RNA polymerase to proceed through and produce the mRNA that then gets converted into the multiple protein products encoded by the lac operon.
* Production of protein is subsequent to the production of the mRNA
* As you add lactose that induces the lac operon and causes lacI to fall off --> RNA polymerase can proceed --> you get the mRNA produced -->a bit later the two proteins are produced
86
Are proteins degraded after their production? If not, what is?
• Proteins- not degraded subsequent to their production but the lac mRNA is
87
When are there lower levels of lac enzymes?
o Only lower levels of enzymes when cell divides and operon isn’t turned on
88
Why is there always a bit of basal expression of the lac operon?
• Always a bit of basal expression of lac operon because the lacI protein isn’t always bound to the operator in the absence of lactose. It is in equilibrium -> goes on and off of the operator DNA which allows a little bit of lac operon mRNA to be produced which enables a tiny bit of that lacY protein to be present which allows a small amount of lactose to get in.
89
How does E.Coli. mediate the preference for metabolizing glucose over lactose?
• This is how E.Coli mediate the preference for metabolizing glucose over lactose
o Does that through the production of molecule called cyclic AMP
• When glucose is high- cAMP is low
• When glucose is low- cAMP is high
• When cAMP is at low glucose levels, binds another protein present in the cell called CAP to induce transcription of catabolic operons like lac
90
What is cyclic AMP?
A modified nucleotide
91
In the lac operon, what is needed in order to recruit RNA polymerase efficiently?
In order to recruit RNA polymerase very efficiently, the CAP binding protein is needed
92
What happens to lac gene expression under different glucose and lactose conditions?
* Lactose available AND low glucose -> high cAMP which binds to CAP protein which recruits RNA polymerase-> Iac genes strong expressed
* High glucose AND lactose unavailable- Iac genes not expressed
* Low glucose AND lactose unavailable-Iac genes not expressed
* High glucose AND lactose available -> low cAMP because glucose is high -> CAP protein isn’t able to bind to the operator-> very low (basal) level of gene expression
93
What kind of system is the Trp operon system?
Repressor type system that has biosynthetic/anabolic set of enzymes
94
What happens in the absence of Trp?
In absence of Trp, trp does nothing and doesn’t bind to the operator and mRNA is produced as normal
95
What happens when Trp is present?
If Trp in its surroundings, the bacterium doesn’t want to make Trp (most complicated amino acid to produce) -> waste of energy.
• When Trp is present, it binds to this trp repressor protein which enables the binding of the Trp to the operator which stop RNA polymerase from binding to the operator -> no production of the mRNA
96
Why is Trp important?
Trp essential amino acid for protein production even though it’s the least common amino acid in proteins
97
The cell doesn't want to completely shut down trp synthesis when there's only a small amunt of trp present in the cell. How does it regulate this?
o trpL encodes a small protein that uses the tight coupling between transcription and translation to regulate the activation of the operon
o TrpL protein produced for regulatory means
o Trp is not bound and RNA polymerase has gone through and started to transcribe the mRNA of this trpL
That mRNA has some specific amino acids encoded in it that, including trp codons, that the ribosome as its going through and translating that mRNA can sense whether there’s enough trp in the cell to synthesise these two side by side tryptophan codons or not
98
What happens at high levels of tryptophan in terms of the attenuation mechanism?
Ribosome moves through the two codons in the trpl sequence and translates them very quickly –lots of tryptophan bound to the tRNA so ribosome is able to incorporate that very quickly into a leader peptide
However, does this with such a rate that it enables the downstream segments (3rd and 4th regions) in the mRNA which have complementary sequences to hybridize and form a hairpin loop tightly evolved with speed of ribosome
Formation of hairpin loop stops RNA polymerase from proceeding down the trp operon and that mRNA is therefore just a truncated mRNA that is produced (attenuated mRNA)
3rd and 4th come together shuts down mRNA synthesis
99
What happens at low levels of tryptophan in terms of the attenuation mechanism?
Low levels of trp tRNA that the ribosome can use the ribosome will stall during trp codon one
• Ribosome has error correction mechanisms can tell if tRNA is present, rightly bound…
This allows 2nd and 3rd elements within the mRNA to hybridise
• 2nd and 3rd come together mRNA synthesis continues on and will produce mRNA encoding all the structural genes which then gets translated into the protein
100
What is vibrio fischeri?
Luminescent bacteria
101
Who does Vibrio fischeri have a relationship with and why does it have a relationship with it?
o Symbiotic relationship under body of bobtail squid
| o Reduces squid shadow makes squid invisible from below during daytime in ocean
102
Why and how does the bobtail squid control bacteria levels?
o During nighttime, bobtail squid doesn’t want the light
Therefore, wants to control bacteria levels at different times
Doesn’t want to lose all bacteria
o Squid controls levels of bacteria present in light-producing organ
o Bacteria can detect cell density in order to control its light production
o When the squid wants light to be produced, it allows the bacteria to get to a very high cell density within this organ squid actively feeding the bacteria with amino acids and glucose in this sac
o When squid doesn’t want area to be lit anymore, can eject the bacterium which eliminates quorum sensing as ejected most of the bacteria
103
What is quorum sensing?
Bacteria can detect cell density of itself and other bacteria in enclosed area
104
In quorum sensing, what is transcription regulated by?
Cell density
105
What does LuxI do and what medium is it useless in in the Lux operon of Vibrio fischeri?
o Produces inducer molecule at constant rate which can freely diffuse in and out of the medium
o No quorum sensing induced in the ocean as molecule just diffuses away
106
What does LuxR do and when is it active in the Lux operon of Vibrio fischeri?
o Can bind to inducer molecule and regulate lux operon
o High cell density + contained environment (such as the squid sac)
--> Contains secreted inducer molecules --> allows concentration build up to a certain level --> once concentration reaches a certain critical level, able to effectively engage the lux R transcriptional regulator protein and induce the luxICDABE genes which ultimately lead to light production
107
Why does Vibrio Cholerae use quorum sensing?
o Quorum sensing to control virulence genes in gut
o Needs to control the production of its virulence genes depending on the cell density of its cell density and other species cell density
108
How does Vibrio Cholerae use quorum sensing?
CqsA detects itself whilst LuxPQ detects other species of bacteria
• Senses its purity in the population
• Depending upon those signals, can induce production of antibacterial inhibitory molecules to fight off the other bacteria or can regulate expression of its virulence genes
109
What cell density does vibrio cholerae want and why?
Wants low cell density to express virulence genes so that the immune system isn’t activated due to the low cell density, which enables the cholera bacteria to produce their toxin and attach to intestinal wall and start to form plaque
110
What happens at high cell density in vibrio cholerae?
At high cell density, virulence genes repressed
• High density of molecules binds to the surface receptors, changes their activity which in turn turns off quorum sensing regulation and turns off virulence genes
• Also produces protease which cleaves plaques off intestinal wall and allows bacteria to go into diarrhea and be spread to other organisms
• Quorum sensing is key to the transmission of cholera to other organisms
111
How does quorum sensing generally work?
o Occurs essentially in all bacteria
o Synthesis of unique metabolite by each bacteria in population at a constant rate
o Bacteria evolves different sensing mechanisms to detect the secreted metabolites with cell surface sensors or intracellular regulators which can bind to a molecule and activate transcription
Can only do this when molecule is at a certain concentration
o High molecule concentration in an enclosed space activates translation to mediate phenotypes
aka
- Synthesis
- Recognition
- Response
112
What are different quorum sensing secreted chemical signals?
o Peptides such as oligopeptides:
In Gram-positive bacteria
Species with this molecules can only detect itself- species specific
o N-acyl homoserine lactone (AHL)
In Gram-negative bacteria e.g. vibrio fischeri
Molecules easily permeate the membrane
Species with this molecule can only detect itself-species specific
o Autoinducer-2
In Interspecies “cross-talk”
Many organisms have it
Used to detect other bacteria present in vicinity
113
What phenotypes are regulated by quorum sensing?
- Antibioic production
- Bioluminescence
- Biofilm formation
- Virulence factors
114
Does protein or mRNA reach a steady state first?
mRNA
115
What influences how genes are expressed?
Signals, proteins and transcription factors
116
What is the activator transcription factor pattern and what is its rate of production?
• Transcription factor: Activator
o X Y
Where X is the activator for the expression of Y
• No X (no activator binding to the promotor) then there’ll be no transcription of gene Y.
Rate of production of Y = f(X*)= EX*n/Kn + X*n –this is the Hill function
• E is the maximum that it gets to
• n>1 Hill functions can be caused by polymerization or multiple small molecules binding makes it more or less active n changes
117
What is the repressor transcription factor and what is its rate?
• Transcription factor: repressor:
o X--| Y
Stops transcription
If not around, you get transcription and translation
Rate of production of Y =f(X*) = Emax/Kn +X*n
Not much repressor -> lots of expression
118
What is basal expression rate and why does it occur?
• Y = E0 + (EX*n)/Kn + X*n
o This formula is for the activator
o Emax is E0
• Often there is small amount of leakage in gene expression
119
What can the Hill function be estimated by and how do you do this?
• Hill functions can often be approximated by logistic (gene switched on/off) functions
• When amount of activator is below K threshold, you approximate it being switched off, when above approximate switch on
o Works extremely well when there is a steeper curve
• Can also get this with 2 transcription factors-
o When protein is bound and the repressor protein not bound- when you get that case then you get the expression
120
Can logistic function with several inputs occur? If yes, describe this.
• Logic functions with several inputs can occur
o cAMP and IPTG
They bind to the same location
o With point mutations, can get completely different shape –looks more like an orb
If there’s cAMP there, switches on
If there’s IPTG, switches on
If there’s both of them, it’s roughly the same
o With another point mutation-
If you add IPTG, practically nothing happens
If you add cAMP, not much happens
You put both of them in, you get gene expression
121
What is response time determined by protein removal rate?
• Response time determined by protein removal rate:
| o 0 -->X : reaction rate =E
122
What is the reaction rate and formula of removal composed of degradation and dilution?
• Removal composed of degradation and dilution:
| o X -> 0: reaction rate = yX
123
What is the response time of stable protein is one cell generation?
• Response time of stable protein is one cell generation
o dX/dt =E –yX
o Completely determined by protein degredation and dilution
o Dependent on kinetic constant associated with the removal rate
124
Why are we not worried about mRNA dynamics?
• mRNA has a short half-life
o Not worried about the mRNA dynamics -> takes not a lot of time
• As transcription changes, time is much faster for higher degradation, while lower degradation takes a lot more time
125
What is autoregulation?
feedback via repression of own expression
126
What is negative autoregulation?
• Negative autoregulation -> repression of a protein on itself
o A type of negative feedback
o As its amount increases, it represses itself
o Occurs very often in Ecoli
o Autoregulation is widespread
127
Why does regulation of a gene occur?
• This occurs because it can speed things up
o Gene in steady state will have much more activity
o Normally, a gene with a lot of promotor activity would increase in activity uncontrollably
o When protein at steady state, negative feedback will kick in and slows it down, and settles down to the steady state much faster
• Also occurs to counteract changes
o Negative autoregulation counteracts changes
o Similar to inhibition of metabolic pathways
o If something’s causing too much expression, then it can be reduced
o If something’s not causing enough expression, can be increased
o When there is feedback less variation system will become a lot more robust
128
What is a feedforward loop?
The FFL, a three-gene pattern, is composed of two input transcription factors, one of which regulates the other, both jointly regulating a target gene.
129
What is a coherent feedforward loop?
o Type that comes up the most
o Allows a lag to occur before Z is switched on- if there’s a signal and a molecule comes, there’s a lag before Z is switched on. But if this disappears, there’s no lag to it switching on
o If nutrients appear for a short period of time, X will be switched on but the lag would make sure that the gene Z, to produce the protein, will not be switched on as there wouldn’t be enough time to metabolize them
o If nutrients appear for a longer period of time, then Z would switch on- as soon as the nutrients disappear, it switches off again
130
Why do coherent feedback loops occur?
o A cell would want this to occur because if there are a lot of short bursts going on, then it’s a mean of regulating its protein production so as not to waste any energy making a protein that’s not going to be useful. Same reason for the sudden switching off.
131
What causes the lags in coherent feedback loops?
o Need a signal from both X and Y to switch on (causes the lag). As soon as signal disappears, X switches off which causes an immediate switch off of Z.
132
What is an incoherent feedforward loop?
In this loop the activator X
activates Z. the activator X also functions to activate Y which is a repressor of Z.
Thus the two paths of the ICFFL act in opposite directions- the direct path
activates Z and the indirect path represses Z. the gene Z increases its
expression levels (high) when it is bound to the activator X*
.The expression of Z
becomes weaker when it is bound to the repressor Y^
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Why do incoherent feedforward loops occur?
o This occurs because
Enzymes can build up a lot faster- faster initiation
Can get a pulse type effect
o An enzyme needs to be around for a very short period, so does this to get a lot of enzyme out in the cell very quickly but doesn’t stick around for long- only needed for the pulse
134
What is specificity in regards to signal molecules?
Signal molecule fits binding site on its complementary receptor- other signals do not fit
135
What is enzyme amplification?
When enzymes activate enzymes, the number of affected molecules increases geometrically in an enzyme cascade
136
What is protein modularity?
Proteins with multivalent affinities form diverse signaling complexes from interchangeable parts. Phosphorylation provides reversible points of interaction
137
What is receptors desensitization/adaptation?
Receptor activation triggers a feedback circuit that shuts off the receptor or removes it from the cell surface
138
What is signal integration?
When two signals have opposite effects on a metabolic characteristic such as the concentration of a second messenger X, or the membrane potential Vm, the regulatory outcome results from the integrated input from both receptors
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What is enzyme localised response?
When an enzyme that destroys an intracellular message is clustered with the message producer, the message is degraded before it can diffuse to distant points, so the response is only local and brief
140
What is phosphorylation?
The covalent addition of a phosphate group to a molecule
141
Why is phosphorylation important?
• Phosphorylation is an important mechanism by which the activity of proteins can be altered after they are formed.
142
How can the activity of proteins be altered after they are formed?
o A phosphate group is added to a protein by specific enzymes called kinases
o This phosphate group is usually provided by ATP, the energy carrier of the cell
o The addition of a phosphate molecule to a polar alkyl group of an amino acid residue can turn a hydrophobic portion of a protein into a hydrophilic and polar portion, thus inducing a conformational change in the structure of the protein
143
What is phosphorylation important for?
o Mediation of the working and inhibition of many enzymes
o Protein degradation
o Transport, control and efficiency during glycolysis
o Several cellular processes and has special significance in biochemistry of living organisms
144
How does cell signaling work?
* Cells have very specific sets of receptors: different receptors sense different things
* Sensory input receptor turned on signal transduction pathway engages in biology forms appropriate response
145
What are receptors?
Transmembrane proteins that bind to signaling molecules outside the celland subsequently transmit the signal through a sequence of molecular switches to internal signaling pathways to initiate a physiological response
146
Are receptors specific?
Yes
147
What are the most commonly phosphorylated amino acids in eukaryotic cells and why?
• Most commonly phosphorylated amino acids in eukaryotic cells are serine, threonine and tyrosine.
o Hydroxyl group gives opportunity for a stable phosphoester
148
What do kinases do?
• Enzymes called kinases catalyze the transfer of phosphoryl groups to organic molecules
The chemical activity of a kinase involves transferring a phosphate group from a nucleoside triphosphate (usually ATP) and covalently attaching it to specific amino acids with a free hydroxyl group. Most kinases act on both serine and threonine (serine/threonine kinases), others act on tyrosine (tyrosine kinases), and a number act on all three (dual-specificity kinases).
149
Why are kinases important?
• Kinases are particularly prominent in signal transduction and co-ordination of complex functions such as the cell cycle
o Phosphorylation and dephosphorylation can impact enzymatic activity, essentially acting like an switch, turning it on and off in a regulated manner
o Phosphorylation of a protein can facilitate binding to a partner protein and hence regulate protein-protein interactions
o Phosphorylation of a protein can also target it for degradation and removal from the cell by the ubiquitin-proteasome system
o Protein phosphorylation has a viral role in intracellular signal transduction: many of the proteins that make up a signaling pathway are kinases, from the tyrosine kinase receptors at the cell surface to downstream effector proteins, many of which are serine/threonine kinases
o Henceforth, ligand binding at the cell surface establishes a phosphorylation cascade, with the phosphorylation and activation of 1 protein stimulating the phosphorylation of another, subsequently amplifying a signal and transmitting it through the cell- the signal continues to propagate until it is switched off b the action of a phosphate
-> they induce signal
150
How many genes encode for protein kinases?
500
151
What amino acids does protein kinases distinguish against and why?
• Technically, each protein kinase can have a different role as they have the ability to add phosphate to any specific proteins
o Protein kinases do not distinguish between serine and threonine, but do distinguish between these two and tyrosine (due to the aromatic ring in tyrosine)
152
What do different sensors that react to different signals all have, and what is the evidence for this?
• Different sensors that react to different signals can all have a kinase base to them inside the phospholipid
o Only respond to certain molecules response only expressed in target cells
Experimental evidence-
• SDS page- separates proteins by size
• Used IRS1 substrate
• First row from the ladder:
o Basal: put nothing in it
o Added insulin to the protein
o PDGF receptor (antibody that is a cell surface tyrosine kinase receptor)
• As can be seen from the experiment, when added insulin, the protein got heavier- (molecular weight of insulin receptor 95,000 Da) suggesting that the receptor was turned on
• Also proved that the sensors were on the plasma membrane
153
What is the role of tyrosine phosphorylation in insulin pathway?
* Insulin activates the insulin receptor tyrosine kinase, which phosphorylates and recruits different substrate adaptors such as the IRS family of proteins
* Tyrosine phosphorylated IRS then displays binding sites for numerous signaling partners
* Insulin signaling induces fatty acid and cholesterol synthesis
* Phosphorylation changes activity of proteins
154
What is the SH2 protein domain
SH2 domains allow proteins to dock to phosphorylated tyrosine residues on other proteins
155
Why is it important that most receptors are in the membrane?
o To turn on, they have to live where their signal lives
o For example, if Pl3K is in the cytosol of the cell, it is not exposed to the substrate and is turned off
o In many cases, substrate of the proteins are located in the phospholipid membrane
o By putting proteins at the membrane, they have access to their substrate
o Because membrane receptors interact with both extracellular signals and molecules within the cell, they permit signaling molecules to affect cell function without actually entering the cell. This is important because most signaling molecules are either too big or too charged to cross a cell’s plasma membrane
156
Why are some receptors not at the surface of the membrane?
o However, not all receptors exist in the exterior of the cell- some exist deep inside the cell, or even in the nucleus
These receptors typically bind to molecules that can pass through the plasma membrane.
157
What is GFP and what can it be used for? Provide an example.
• Green fluorescence protein
• Can be attached to molecules or enzymes to show their activity
o E.g. to show effector protein recruitment
Recruitment necessary to increase the chance that two proteins will interact with each other- often recruited to at the plasma membrane
• E.g. Akt has to be phosphorylated by PDK1 and PDK2 so the fact that they’re sucked up increases the chance that they’re going to interact with each other
158
In signaling, R+Rp=Rptotal. Why?
Because total amount of protein is constant
159
Using mass action, how can the signal that works on R and Rp be modelled?
o dRp/dt =k1S(Rptot-Rp) –k2Rp
160
What can signals be?
o Could be osmotic pressure
o Small molecule binds to sensor kinase
As it binds, makes sensor kinase change conformation
Makes kinase domain phosphorylated
161
What can a response regulator be?
o Can be an activator or repressor
o Can regulate gene regulation
o Makes enzymes active
162
How are kinases regulated?
Because protein kinases have profound effects on a cell, their activity is highly regulated. Kinases are turned on or off by phosphorylation (sometimes by the kinase itself - cis-phosphorylation/autophosphorylation), by binding of activator proteins or inhibitor proteins, or small molecules, or by controlling their location in the cell relative to their substrates.
163
What does the bifunctional signalling variant do?
You get double the effect
164
How does the MAPK signaling cascade work?
- Widespread in eukaryotes
- Classical mitogen-activated protein kinase (MAPK) pathway activation begins at the cell membrane, where small GTPases and various protein kinases phosphorylate and activate MAPKKKs.
- Subsequently, MAPKKKs directly phosphorylate MAPKKs, which, once activated, phosphorylate MAPKs.
- Activated MAPKs interact with and phosphorylate numerous cytoplasmic substrates and ultimately modulate transcription facts that drive context-specific gene expression
The signal starts when a signaling molecule binds to the receptor on the cell surface and ends when the DNA in the nucleus expresses a protein and produces some change in the cell, such as cell division. The pathway includes many proteins, including MAPK (mitogen-activated protein kinases) which communicate by adding phosphate groups to a neighboring protein, which acts as an "on" or "off" switch.
165
What are MAPK signaling pathways essential for?
```
o Inflammation
o Cell stress response
o Cell differentiation
o Cell division
o Cell proliferation
o Metabolism
o Motility
o Apoptosis
```
166
What is the MAPK pathway for?
The MAPK pathway is a chain of proteins in the cell that communicates a signal from a receptor on the surface of the cell to the DNA in the nucleus of the cell.
-Chain phosphorylation
167
What do the reactions from MAPK to MAPK-p, and from MAPK-p and MAPK-pp have in common? What does this do?
• V1 and v3 have same enzymes, so saturation occurs on both reactions – reaction rate is at steady state (steady state= rate of flux=0)
168
What do the reactions from MAPK-pp to MAPK-p, and from MAPK-p and MAPK have in common? What does this do?
o If enzyme saturated from v4, v2 won’t get a lot of reaction as both use the same enzyme
169
What does equilibrium depend on?
• Equilibrium can depend upon initial condition:
o The system has memory
o Where it settles down to steady state depends on its initial condition
o A system can have multiple equilibrium points
For number of equilibrium points, look if the graphs are overlapping at a particular value on the x axis
170
What is perfect adaptation?
- In cell signaling, adaptation can be defined as a process where a system initially responds to a stimulus, but then returns to basal or near-basal levels of activity after some period of time.
- Adaptation limits the duration of a response and allows for the basal activity of the system to be homeostatically maintained
* No matter what S is, it will eventually settle down where only the kinetic values matter
* In steady state, response is independent of S
171
Describe perfect adaptation before and after in bacteria
• When in a pipette + test tube + nutrients-
o Bacteria start moving towards end of pipette -> swim from area of low concentration to high concentration
If chemotactic receptors at end of the cell don’t sense anything,sends signals that makes flagella move clockwise, causing the tumble and then swim in the new random new direction
o If nutrients are higher in the direction they are swimming, will keep swimming there
o Otherwise, they will tumble until they find a new direction to swim in and attempt that direction
o Essentially tests level of nutrient concentration by swimming
• If the nutrient level increases everywhere, the perfect adaptation mechanism kicks in
o Signaling system adapts and adjusts to a new baseline
o After initial response to some external stimulus, bacteria tumbling frequency often reverts to its original value with high accuracy, independent of the strength of the external stimulus
172
What is the receptor mechanism involved in bacterial perfect adaptation
1. Nutrients act as attractant
2. Toxic substances act as a repellant
3. MCP receptor able to detect attractants and repellants
4. Ligand activates MCP , which recruits CheW (transducer protein) and CheA (sensory kinase- auto phosphorylates itself)
5. CheA protein activates CheY protein, which phosphorylates flagellar motor switch and induces tumbling
• Phosphorylation at Y determines tumbling frequency
• Methylation of WA sensor- if methylated, won’t phosphorylate Y
173
Is perfect adaptation robust?
Yes, it doesn't matter what initial conditions are
174
Describe how adaptation in E.Coli occurs. Draw the diagram.
* Attractant- Signal R -> tumbles in response, which goes on to phosphorylate Y
* Straight line- -> when the bacteria swims up the gradient
* Adaptation in E.Coli is based on reversible methylation and demethylation of receptors at specific modification sites, catalyzed by enzymes CHeR and phosphorylated CheB respectively
* Transmembrane chemoreceptors have major modification sites: receptor modification regulates the receptor activity and provides a recording of experienced concentration changes, and the rate of tumbling was found to adapt precisely for different ligand concentrations
* To achieve the return of the receptor activity to this pre-stimulus value, receptor activity dependent phosphorylation of CheB provides a negative feedback on the receptor activity
* In addition, the rates of methylation and demethylation depend on the receptor activity
Look at the diagram
175
Draw mathematical model types
Draw them
176
Draw biological model types/ process
See lecture slides
177
What is km?
The concentration of substrate which permits the enzyme to achieve half Vmax
