3. DNA, transcription, and networks Flashcards
1
Q
How many dna bps per turn
A
10
2
Q
DNA bp spacing
A
0.34nm
3
Q
How many H bonds between bps?
A
3 for C-G, 2 for A-T
4
Q
DNA helix diameter
A
2.4 nm
5
Q
vertical size of major and minor grove
A
major = 1.2 nm
minor = 0.6 nm
6
Q
Name the 6 stages of DNA compaction
A
- DNA helix
- Beads on a string
- chromatin fibre of packed nucleosomes
- Extended section of chromosome
- Condensed section of chromosome
- Entire mitotic chromosome
7
Q
Describe the strucutre of a nucleosome
A
A histone core wrapped in DNA joined to other nucleosomes by linker DNA
8
Q
How many times does DNA wrap around histone?
A
About 1,7
9
Q
How many contacts between dna and histone?
A
14
10
Q
Energy cost is per contact is
A
6kT
11
Q
Why must nucleosomes be accessible (4)?
A
- DNA repair
- DNA replication
- Gene transcription
- Transcription regulation
12
Q
How does RNAP deal with nucleosomes?
A
DNA that the RNAP has already passed loops round and ‘hugs’ the histone. The histone is then transferred to this section of DNA
13
Q
Derive the kinetics of protein binding to DNA on a nucleleosome. What is the problem with this model?
A
Problem = hard to measure k_d_apparent
14
Q
How can DNA cutting enzymes be used to probe nucleosome dynamics?
A
If DNA can be unwound we would expect cutting to occur in all locations but most commonly away from the centre of each section as this would involve the most unwrapping. This is studied using gels - large sections of DNA move slower.
15
Q
How can nucleosome dynamics be studied using optical traps?
A
One end of DNA is attached to a bead in a trap, the other is attached to a bead attached to a micropippete. Micropippete is moved by piezeelectric actuator, force required to keep other bead stationary in optical trap is recorded. Graph shows to regions: first a reversible unfolding followed by an irreversible detachement fo teh histone form the DNA.
16
Q
How many base pairs in a bacteriophage?
A
~50,000
17
Q
Size of bacteriophage
A
~27nm diameter
18
Q
Packing ratio is?
A
number of base pairs/volume in nm^3
19
Q
Derive the elastic energy needed to pack DNA in a bacteriophage
A
See pages 33-35
20
Q
Derive the electrostatic energy packing DNA in a bacteriophage
A
21
Q
How can optical tweezers be used to study DNA packaging in bacteriophages?
A
A bead in an optical trap is attached to one end of the DNA whilst the other lies within the capside. A specific antibody links the capsid to a second bead. ATP is added. Two options follow: use a force clamp and measure packaging or run no feedback and measure the stall force.
Force clamp shows that the packaging rate decreases rapidly after 50% of the DNA is packed. No feedback shows a strong force (~60pN), reveals two bps are packaged per ATP and that teh force increases as the genome is packed.
22
Q
Half life of mRNA
A
~ a few minutes
23
Q
riboswitches =
A
untranslated pieces of mRNA. They change conformation when bound to small molecules and can thus modulate gene expression by altering the transcription efficiency.
24
Q
Desccribe the bacterial transcritpion cycle
A
- RNAp aseemles with a sigma factor to form RNAP holoenzyme
- RNAP unsiwinds the DNA at the point where transcription begins
- Transcritpion begins. Initially it is inefficient but after about 10bp RNAP detaches from the promoter and weakens its connection to the sigma.
- RNAP switches to elongation mode
- A termination signal is reached and transctiption stops
25
Which direction does RNAp work in?
5' -\> 3'
26
Which protein recognises promotor regions?
Sigma factors
27
Name the two critical steps before transcription can begin
1. Search for promoter region
2. Open complex formation
28
Derive k for the search for a promoter region by 3D diffusion
29
Sketch a transcription complex
30
What is the minimum distance between transcritption bubbles?
It takes 2 seconds for an RNAp to bind. The minumum separation is
size of RNAP + time to bind \* speed to RNAP = 80 +2x50 = 180bp
31
How can the speed of RNAP be measured?
Attach DNA between two beads in optical traps. Add a fluroescent RNAp and monitor its process.
32
How can transctiption elongation be studied using optical tweezers?
Attach DNA between two beads in optical traps. Make one bead oscillate. Then introduce a third bead decorated with RNAp. When an RNAp binds the second bead will no longer oscillate as their motion is no longer coupled. To analyse transcription elongation, monitor the bead downstream of the RNAp.
33
How can FRET be used to study transcription initiation?
A FRET pair is introduced at specific points on the DNA. RNAp is then introduced. RNAp will alter the separation of the FRET pair as it passes between them, altering the FRET efficiency.
34
How is the position of the magnetic bead monitored in a magnetic tweezers experiment?
By analysing its interference pattern.
35
What is the linking number?
twist + writhe (supercoils)
36
By how much does using supercoils amplify the signal
One turn of unwinding becones a 56nm change when converted to a superoil
37
What can be learn about transcription from a magnetic tweezers experiment (4)?
1. The number of unique states - given by the number of different extensions observed.
2. The changes in DNA extension.
3. The waiting time for the open complex to form once RNAP has been introduced and its dependence on RNAP concentration.
4. The waiting time for the open complex to close. This is independent of RNAP concentration.
38
What are the four discrete steps in process of creating a transcription complex observed using magnetic tweezers?
intital state, elongation complex, open complex, initial transcribing complex
39
What is abortive initiation?
An early process of genetic transcription in which RNA polymerase binds to a DNA promoter and enters into cycles of synthesis of short mRNA transcripts which are released before the formation of the elongation complex
40
Why is abortive initaition important (3)?
1. Regualtion - it can be rate limiting
2. Allows promoter escape
3. Some antibiotics work by blocking this process
41
3 models of abortive initation and which is correct and why?
1. RNAp inchworming - implies there is a flexible element in RNAp
2. DNA scrunching - implies that DNA is flexed, resulting in aditional unwinding during initiation
3. Transient excursions - reversible RNAp translocation on DNA
2 is correct - the initial transcribing complex has a differnent lenght to the elongation complex
42
4 ways of studying elongation
a) study Brownian motion as tether changes length
b) study force/displacement
c) study separation of beads
d) study force/displacement/velocity
43
What evidence is there that the rate limiting step of elongation in biochemical (2)?
On a force velocity plot
1. velocity plateaus at low force
2. force is very large
44
What are the two models of transcription by RNAp and how can they be distinguished?
1. Powerstroke - suggestes there is a conformational change in the RNAp betwen relaxed and stressed states.
2. Brownian ratchet - based off thermal flucutation between states with different binding affiniting for incoming NTP
Can be distinguished by the distance over which the force acts - for a powerstroke model the force acts over only part of 0.34nm whereas in the brownian ratchet model it acts over the full 0.34nm
45
How can single base pair resolution be achieved in an elongation experiment (3)?
1. Levitation - decouples system from stage, reducing noise. Acheived by holding all beads in optical traps.
2. Helium - has a lower RI that air so reduces photon depletion.
3. Hold the bead attached to DNA in a strong trap and the one attached to RNAp in a weak trap
46
What is the effect of a brownian ratchet model with multiple binding sites?
High sensitiivty to force at high ATP concentration
47
How can the rate of protein production in a cell be monitored?
By engineering the DNA to produce proteins tagged with flurescent proteins.
48
Repressor =
a protein which binds to DNA to block RNAP from binding to the promoter region
49
operator =
the region of DNA where repressors bind
50
activator =
a protein which binds to DNA to increase the rate of formation of the RNA-DNA open complex through interactions with RNAP
51
Where do activators bind?
upstream of the promoter region
52
Explain the lac operon
Cells prefer to use glucose to lactose, but in the absence of glucose, will produce lactase to break down lactose.
If lactose and glucose are both present, neither activators nor promorors will bind. Lactase will not be produced
If lactose is present and glucose is not present, activators will bind. Lactase will be produced.
If lactose is not present, repressors will bind to the operator.
53
What are the repressor and activator in the lac operon called?
repressor - lacl
activator - CRP
54
Describe one model of how lacl works?
It twists the DNA, preventing RNAP from binding and sliding
55
Find the 3D search time for RNAP finding its target, in terms of the distance to the target and diffusion constant
56
Find the 1D sliding distance in terms of D1 and k\_off when RNAP is searching for a promotor region
57
Find the time taken for RNAP to reach a promotor region, given
t\_1D = l\_sliding^2/D\_1
t\_3d = r\_target^2 /D\_3
58
What is the effect of changing the sliding lenght?
Increasing the sliding lenght essentially increases the reaction radius ie a protein can bind far from the binding site and slide into place.
However if the sliding length is too long, too much time will be spent exploring DNA far from the binding site.
59
What are the two paths a lambda bacteriophage can follow after infecting a bacterial cell
Lysogeny - the lamda DNA replicates alongside the host chromosome. The host is not harmed.
Lysis - the bacteriophage replicates rapidly before bursting out.
60
What are the four steps involved in lysis?
1. Proteins N and cro are expressed.
2. Protein N helps the
1. trascription of genes associated with creating proteins that form part of the bacteriophage
2. replicating DNA to fill the new bacteriophages
3. These parts are expressed and assembled
1. They break through the host membrane
61
What is the order of priority of the cl repressor?
1. Stop transcritption of lamda cro
2. Activate transcritption of itself
3. Repress transcritpion of itslef
62
How does cl concentration determine whether lysis will occur?
High cl concentration - lamda cro is not transcribed and cl is kept in a steady state. Lysogeny occurs
Low cl concentration - lamda cro is activated and the process of lysis begins.
63
Transcription factors =
Proteins that represent the environmental state. They can transit between active and inactive states rapidly.
64
signal =
Tthe input to a transcription network. Causes a change to the physical shape of a transctoption factor which transitions it to the active state ie the signal S causes the inactive X to become the active X\*
65
Illustrate transcription with no activator or repressor
66
Illustrate transcription with an activator
67
Illustrate transcription with a repressor
68
How long does it take for a signal to bind to a transcription factor?
~1ms
69
How long does it take an active transcription factor to bind to DNA?
~1s
70
How long does the transcription and translation of a gene take?
~5 mins
71
How long does it take for a 50% change in protein concentration?
~1 hour
72
Write down the Hill function and label its variables
73
What is the activation coefficient in the Hill funciton?
The concentration of X\* that is required to have a signicant effect on transcription of Y.
74
Write down the Hill function for an activator and label its variables
Beta = max expression
K = activation coeffiecient
75
What is required for a motif to act like a switch?
It must have a point of inflection
76
How can K be changed?
By altering the DNA sequence at the binding site of X\*
77
How can beta be changed?
By mutating the RNAP binding site
78
What two factors make up the total degradation rate?
Protein degradation and dilution due to cell division
79
Response time =
The time taken to reach halfway beween the initial and final states. For simple growth and decay t\_1/2 = ln2/alpha
80
network motifs =
Patterns that occur more frquently in real networks than randomised networks. They can be used as building blocks to construct more complex networks.
81
Autoregulation =
regilation of a gene by its own gene product. Represented by a self-edge
82
What are the benefits of negative autoregulation?
Reduces the response time.
More resistant to fluctuations in beta since the steady state value is only dependent on K, which varies much less from cell to cell.
83
When is positive autoregulation useful (2)?
Processes which take a long time eg developmental processes - positive autoregulation increases the response time.
Irreversible decisions eg what type of tissue a cell will become - when alpha is small positively autoregulated systems can be bistable
84
Differnce between coherent and incoherent feed forward loop?
In a coherent ffl the indirect path has the same overall sign as the direct path, in an incoherent ffl the signs are opposite.
85
Why are type 1 coherent ffl useful?
Using AND logic - acts as a persistance detector - if s\_x is on for a short time Z will not be produced because tehre is a delay during the on step but not during the off step. Useful for sugar break down eg in ecoli AND NOT logic is used to determine whether to break down glucose or arabinose.
Using OR logic - acts the opposite way around. Useful for flagella motor
86
Repression factor =
beta\_z/beta\_z'
where b\_z corresponds to YK\_yz.
Increasing therepression factor increases the pulse like dynamic of Z production
87
When is an incoherent type 1 ffl with AND logic useful (2)?
1. To create pulse like behaviour
2. When a response time needs to be quick (note that response time is dependent on the repression factor)
88
Name three ways to reduce response time of a nework
1. Increase the degradation rate alpha
2. Use negative autoregulation
3. Use an incoherent type 1 ffl
89
single input module =
a motif where one regulator controls a group of genes
Note that all regulations signals are the same, each of target genes has only one input and the master transcription factor is usually autoregualted.
90
Functions of single input modules (3)
1. Controlling genes which have a common biological function eg working sequentially to assemble a desired molecule.
2. Controlling groups of genes that respond to a specific stress eg DNA damage, heat shock
3. Controlling groups of genes that together make up a protein machine. The gene products then self aseemble into a molecular machine.
91
When is temporal order important in transcription (3)?
1. Genes that are timed throughout the cell cycle
2. Genes regulated by the time of day
3. Genes involved in developmental processes
92
When is the multi-output ffl useful?
When assembling flagella motor - proteins are encoded on 6 operons.
93
When are oscillatory networks useful (4)?
1. Body clock
2. Heartbeat
3. Spiking neurons
4. developmental processes whcih generate repeating tissue
94
How does an oscillator network work?
There are two time scales - X activales Y and itself on a fast timescale while Y represses X on a slow timescale because Y interacts with X on a protein-protein level.
95
Sketch a repressilator circuit
96
Derive the error rate in translation without kinetic proofreading
97
Derive the error rate in gene expression with kinetic proofreading
98
Derive the rate of transcription without noise
99
Find the steady state protein concentration from the Langevin equations
100
What is the burst size?
The average number of proteins produced per RNA
101
Define the Fano factor
102
On what timescales do RNA and protein degradation occur?
RNA ~ 2mins
Proteins ~ 20 mins
103
What is the differnce between intrinsic and extrinsic noise?
Intrinsic - comes from within cell, doesn't correlate with other cells
Extrinsic - comes from outside cell, correlates with other cells
104
Why are fluctuations important for transcription (3)?
1. They cause mixed populations - different cells have different levels of expression of the target gene. This increases with the length of the cascade.
2. Allow microorganisms to respond to environmental changes and avoid getting trapped in suboptimal epigenetic states.
3. Important for cell differentiation - provide the intital hetergeneity on which cell-specific genes can propagate.
105
Diffusion coefficient =
kT/(6 pi viscosity radius)
106
Sketch the lifecycle of a bacteriophage
