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Heather knight Flashcards

1
Q

Explain the process of elongation

A

RNA polymerase unwinds the DNA as it goes and makes a bubble.

  • Free ribonucleotide triphosphate bind to form the new chain.
  • The polymerase makes sure they are the correct matches using its proof reading function.
  • not as good as DNA polymerase but then it doesn’t need to be
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2
Q

What is the function of RNA polymerase in elongation?

A

It catalyses the formation of phosphodiester bonds that link nucleotides together in a chain.
- same as in DNA

The 5’ end, ends with a nucleotide in which the triphosphate group has not joined in a linkage with to make a phosphodiester bond.

The 3’ OH. Group attaches the 5’ phosphate of new nucleotide triphosphate to allow the chain to grow

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3
Q

What does the inverted repeat sequence of the terminator sequence in termination result in?

A

Mean that the synthesised RNA molecule will pair to itself and form a hairpin structure.

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4
Q

What does the formation of the hairpin structure cause?

A

May help to pull the transcript away from the RNA polymerase’s active site. In bacteria there are 2 types of termination signal.

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5
Q

What happens after coding sequence of a gene is transcribed into mRNA?

A

Transcription must stop. The RNA polymerase will only release the growing chain when it encounters a termination signal.

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6
Q

What structure do the termination signals in bacteria form?

A

Stem and loop, secondary structure through H bonding between Gs and Cs

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7
Q

What characterises intrinsic terminators?

A

A C-G rich stem followed by a run of As in the template strand.

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8
Q

How does the structure of the intrinsic termination sequence aid it’s function?

A

Hairpin structure is very stable.
- this makes it more difficult for DNA RNA base pairing to continue in the in the Hubble and will disrupt the progress of the RNA polymerase along the DNA.

  • in addition when the run of A’s in the terminator sequence is encountered and transcribed by the polymerase, these results in AU pairs with only 2 H bonds compared with 3 for each of the GC pairs in the stem loop.
  • in conclusion the stem loop structure is preferred to the ‘bubble’ do it is hard for the hybrid DNA RNA to stay attached and the RNA chain is released
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9
Q

What is a possible role for the flap structure?

A

RNA hairpin may be in contact with the flap structure.

Movement of the flap may contribute towards the breakage of the RNA-DNA hybrid and the ultimate termination chain.

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10
Q

What are Rho-dependant terminators?

A

These require a protein called Rho for termination of transcription. Rho attaches itself to the transcript as it moves along the RNA toward the polymerase.

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11
Q

How do Rho dependant terminators stop transcription?

A

Rho catches up and uses its activity as a helicase enzyme to break the base pairs between the DNA and RNA, stopping transcription.

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12
Q

Why is it important that the amount of a gene being transcribed varies hugely?

A

Not all proteins are required in the same amounts or at the same time. Switching genes in or off when needed saved energy

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13
Q

Explain the process of transcription initiation step 2

A

Once mRNA has begun to be synthesised, the sigma factor is released from the holoenzyme and the process of RNA polymerase moving down the chain starts in a more efficient manner.

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14
Q

What are the 3 types of transcriptional control mechanisms used by bacteria?

A
  1. Operons: coordinated control of gene groups
  2. Alternative sigma factors: decides which genes are transcribed
  3. Regulating transcription termination: decides whether addition genes are transcribed
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15
Q

What are 2 types of operons and give an example of each?

A

Catabolic (lac operon)
Biosynthetic (Trp operon)

Can be controlled by positive or negative mechanisms

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16
Q

What are a group of genes with related function called?

A

An operon

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17
Q

What is an operon?

A

A group of genes controlled by a shared promotor.

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18
Q

Genes on one operon are transcribed together to form a polycistronic mRNA what is this?

A

mRNA consisting of a continuous transcript that represents several different genes.

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19
Q

What is positive control in relation to onerous?

A

+ve control mean the DNA binding protein binds and switches transcription ON. -ve control is the opposite.

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20
Q

What is catabolism in terms of opera singer?

A

Is breaking down molecules to make something that is needed. Catabolic onerous control the expression of enzymes used in sugar metabolism and utilisation.
- Lactose Oberon is catabolic because they will only express enzymes when they are required I.e. If glucose is not avalible as a food source.

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21
Q

What is the structure of Lactose, why can’t E.coli break it down?

A

It is a disaccharide made up of 2 monosaccharide sugars joined together. These two sugars are glucose and galactose. E.coli can use the monosaccharides but cannot use the disaccharides.

Glucose and galactose are joined together by a 1,4 linkage. Bacteria cannot utilise this sugar so need to split it into its two component parts using an enzyme Called beta galactosidase.

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22
Q

What are the 3 steps called that enable lactose to be utilised?

A
  1. Detection- to see if lactose is avalible outside the cell
  2. Import- to import it into the cell
  3. Cleavage- to cleave into 2 sugars it can use.
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23
Q

Regulation of the lactose Oberon is ‘coordinate’ what does this mean?

A

All the enzymes are regulated in an identical fashion. The three genes lacZ/Y/A constitute an Oberon. They are structural genes and are transcribed from a single promoter into polycistronic mRNA. V rare in eukaryotes.

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24
Q

What are structural genes with regards to opereons?

A

They are concealed with the business of the Oberon, they code for enzymes.
This is in contrast to regulatory genes where they are concerned with the control of the Oberon.

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25
Q

What is special about LacI?

A

It has its own promoter and terminator this showed that it is a regulatory gene because when they disrupted its function by muting it all of the other three genes were expressed all the time.
- when it’s not disrupted we see control of the expression of LacZ T and A under different conditions.

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26
Q

What is the function of LacZ?

A

Codes for the enzyme beta-galactosidase which cleave lactose into galactose and glucose. Glucose is used immediately, when used up galactose induces another different Oberon to then be used up).

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27
Q

What does LacY code for?

A

Lactose per ease which transports lactose into the bacteria as it cannot simply diffuse across the plasma membrane.

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28
Q

What does LacA code for?

A

Transactylase.

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29
Q

What is the function of LacI?

A

Encodes a repressor (tetramer)

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30
Q

What is LacO?

A

Is next to lacZ and is the operator sequence.

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31
Q

When lactose is is not present, what happens?

A

LacI gene is transcribed and produces mRNA that is translated into a protein.

  • 4 copies of this protein aggregate to form a tetramer.
  • the tetramer is a repressor which binds to the operator region in the lac operon’s promoter and stops transcription occurring.
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32
Q

How does steric hindracne prevent the expression of the operon?

A

In the absence of lactose the repressor protein is transcribed (it always is by default). And acting as a tetramer, binds to the operator site. This interferes with the binding of RNA polymerase to the promoter by steric hindrance.

  • the binding sites of RNA polymerase at the promoter and lac repressor at the operator overlap, hence the binding sites of RNA polymerase at the promoter and lac repressor at the operator overlap.
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33
Q

When there is no lactose what happens at the operator site?

A

The repressor protein binds preventing transcription of the Lac operon.

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34
Q

How does lactose induce expression?

A

By inactivating the lac repressor. Inducer converts lac repressor into an inactive form that cannot bind. RNA pol binds at promoter and transcribes the operon.

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35
Q

What effect does permease have in the lac operon?

A

Imports a few molecules of lactose and this is enough to start the next step. The inducer binds to the repressor and causes it to undergo a conformational change so that it can no longer recognise and bind to the base sequence of the operator. Thus RNA polymerase can now get to work to express the operon.

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36
Q

Why is there always some permease and beta-galactosidase present?

A

Everything in biology is in equilibrium- nothin is absolute.

Repressor (unbound) + operator DNA Repressor (bound)* Operator DNA

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37
Q

Lactose is not really the inducer. What is? and how is it produced?

A

beta-galactosidase can produce some side products and rearrange lactose rather than cleave it.

  • one of the side products is allows toes which is beta-galactose 1-6-glucose.
  • It is allolactose that is the inducer, this is present whenever lactose is.
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38
Q

What happens to the repressor molecules when lots of lactose is present?

A

All repressor molecules have bound allolactose.

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39
Q

What happens when the repressor is not bound to the promoter?

A

RNA polymerase DOES have access to the promoter because the repressor has been deactivated due to the binding of allolactose. The 3 structural genes are transcribed and later translated into enzymes.

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40
Q

How did Jacob and Monod gain the proof for the lactose operon?

A

E.coli mutant strains that didn’t normally respond to lactose.

  • the mutations were in the genes that encode the regulatory system of the lac operon.
  • they isolated two types: lacI- and LacOc- mutations
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41
Q

What is characteristic of a LacI mutant?

A

They are constitutive

  • lacZ is always expressed
  • LacI- is a mutant version of the LacI gene that encodes a mutant form of repressor that cannot bind to LacO, so expression is always on. > RNA polymerase can always bind to the promoter.
  • so the constitutive effect is the constitutive expression of LacZ
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42
Q

Look at heather 1/4

A

Pages referring to mutants and their effects read though.

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43
Q

How is the catabolism of glucose able to repress the lactose operon? (I.e. When glucose and lactose is present, because the repressor-allolactose complex will still form allowing RNA polymerase to transcribe)

A

The process takes place involving a small molecule called cyclic AMP.

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44
Q

What does AMP catalyse?

A

The cyclisation of ATP into cyclic AMP.

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45
Q

What effect does high glucose have on cAMP?

A

High glucose means low cAMP

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46
Q

What did E.coli cya mutants show us?

A

They have defects in the enzyme adenyl cyclase and are unable to make cAMP. They are therefore unable to activate any of the ‘sugar operons’.

When treated with cAMP the mutant strains immediately developed operon inducability.

Therefore cAMP must be required for inducability.

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47
Q

What is the name given to the glucose phosphotransferase system?

A

The phosphoenol pyruvate.

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48
Q

How does the glucose phosphotransferase system work?

A

Protein IIA-Glc (is specific to glucose). When glucose is present the transport past these enzymes it becomes dephosphorylatesd. Preventing the conversion of ATP to cAMP. As cAMP is required for transcription of the operon, nothing is transcribed.

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49
Q

How does cAMP aid transcription?

A

It binds to a receptor called CAP (catabolise gene activator protein).
cAMP allows CAP to bind to the CAP site.

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50
Q

If glucose levels are high what happens to cAMP levels, what position is CAP and what is the result with regards to transcription?

A

Glucose high = cAMP low + CAP detached + no transcription of lacZ

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51
Q

When glucose is not present and the IAG1c is phosphorylated what events follow?

A

This form of the protein stimulates adenyl cyclase to produce cAMP and this allows the CAP protein to attach to the lac gene promoter and transcription can occur.

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52
Q

What happenes if both glucose and lactose are available to the cell?

A

CAP is detached and the lac operon is not transcribed even though the presence of lactose means that the repressor has Ben inactivated. The effect of glucose takes precedence over everything.

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53
Q

What is the function of CAP?

A

CAP binds to DNA in the promoter.

- the CAP binding site in the promoter consists of palindromic sequences.

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54
Q

What effect does CAP have on DNA?

A

CAP bends the DNA to force it apparat.

- the CAP protein is required to break the strong bond. It is fairly rigid so effort is needed to get it to bend.

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55
Q

What are the structural genes of the trp operon?

A

It has 5 structural genes, trp E/D/C/B/A that encode the trp repressor.

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56
Q

How do the needs of the trp operon differ to lac operon?

A

They are opposite in function.

E.g. If tryptophan is present in the growth media the bacteria do NOT need to synthesise it.

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57
Q

What effect does tryptophan have on the repressor.

A

It represses the expression of the operon.

  • the repressor by itself is inactive, it is the aporepressor.
  • it must complex with tryptophan itself the corepressor before it is active (the halo repressor).
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58
Q

What effect does the haloporepressor have on the trp operator?

A

It binds to the trp operator (trpO) and inhibits transcription by steric hindrance.

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59
Q

What is negative control with respect to regulation?

What is induction, what is repression?

A

It is a repressor.
Induction = inactivating the repressor
Repression = letting the repressor work

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60
Q

What is positive control with respect to regulation?

  • what does induction do?
  • what does repression do?
A

It acts as an activator

  • induction = means letting the activator work. Activator is present and allowed to work.
  • repression = stopping the activator from working. Lacks activator or a non-functional version that cannot work
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61
Q

What control mechanisms do the lac repressor and trp use?

A

Negative control.
If the lacI and trpR genes are deleted or inactive we get constitutive expression of the operons.
This means that in the absence of a regulatory control the operons are transcribed and translated.
To regulate them we have to switch them off.

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62
Q

If an operon is regulated by positive control what happens if gene for a regualtory protein is inactivated or deleted?

A

The operon will never be expressed.

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63
Q

In the absence of the activator protein in positive regulation what happens?

A

There is no expression at all. Even a low conc of activator is enough to switch on transcription.

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64
Q

What form of regulation of operon is only known to occur in eukaryotes and how does it work?

A

Positive control- repression.

A positive agent binds to the activator and makes it inactive.

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65
Q

What is special about the Arabinose operon?

A

It uses both positive and negative control.
TL-Arabinose is another such which if necessacery can be catabolised if no glucose is avalible.
Arabinose operon is regulated by both positive and negative control and it has 2 regulator DNA sequences: araO and araI.

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66
Q

How is the Arabinose operon controlled?

A

The AraC protein acts as a repressor. It binds to the operator araO and to araI. In doing so causes the DNA to from a loop that results in repression of transcription of the three structural genes (araBAD)
- in the presence of Arabinose itself, Arabinose binds to the AraC protein and converts it to an activator protein. This now binds to at the araI site pans switches on transcription. Like in lac operon the switching on also requires cAMP-CAP.

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67
Q

What are alternative sigma factors as a method of transcription regulation?

A

They decide which genes are transcribed.

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68
Q

Which sigma factor is used to switch on the expression of most ‘housekeeping genes’?

A

The sigma70

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69
Q

What does the sigma70 recognise?

A

-35 box.

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70
Q

What effect having would a different sigma factor have when used by RNA polymerase?

A

It would recognise a different protein. Used in situations for switching on and off different groups of genes in bacteria.

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71
Q

What effect does adding a different sigma factor to the core RNA polymerase enzyme have?

A

It causes a different holoenzyme to be made. These different versions having different sigma factors- will have different recognition capabilities.

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72
Q

What induces the synthesis of alternative sigma factors?

A

Particular conditions, including conditions (such as heat shock) that cause protein miss-folding.

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73
Q

Under heat shock what sigma factor is produced, and what are the differences to sigma 70?

A

Sigma32. It has different recognition properties and doesn’t recognise the consensus promoter sequences instead it recognises different ones. These are found in the promoter of heat shock genes (genes that are required to be switched on specifically to deal with heat shock)

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74
Q

What promoters to the sigma32 recognise?

A

Induced by unfolded proteins in the cytoplasm.
Genes encoding chaperones that re-fold the unfolded proteins and protease systems leading to the degradation of unfolded proteins in the cytoplasm.

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75
Q

What promoters to do the sigma factor sigma54 recognise?

A

Genes for nitrogen metabolism and other functions.

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76
Q

Regulating transcription termination: what is antitermination?

A

It allows terminator signals to be ignored.

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77
Q

How does antitermination work?

A
  • RNA polymerase moves along the promoter and when it reaches antiterminatior protein it picks it up and brings it with it. RNA pol ignores a termination signal and carries on synthesising transcript until a second signal is reached.
  • this is one way of allowing further coding regions to be transcribed.
  • Antitermination is an important part of the infection stratagem of phage lambda.
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78
Q

What controls nearly all amino acid bio synthetic operons?

A

Attenuation.

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79
Q

How can the formation of stem and loop structures alter the termination of gene transcription?

A

You can either have stem-loops 1 and 3, then you make a terminator or you can have stem-loop 2 in which case you do not get a terminator.

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80
Q

What is the name given to the ‘short gene’ that overlaps stem and loops 1 and part of 2 coding for a 14 aa polypeptide?

A

A small open reading frame (ORF)

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81
Q

What formation of stem and loops always stops transcription?

A

Unless prevented by a mechanism the stem and loops will dorm in the order in which they are transcribed, so stem and loop 1 forms first, which precludes stem and loop 2 thereby allowing stem and loop 3 to form.

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82
Q

What happens to transcription/translation with high Trp?

A

Peptide is translated. Ribosome dissociates.
The end of ORF is before the second stem and loop structure is completely transcribed.
- so once the ribosome dissociates the first stem and loop strucutre will form, leading eventually it the formation of the 3rd stem and loop and resulting in transcription termination.

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83
Q

What happens to transcription/translation when there is low Trp?

A

Under reduced tryptophan concentrations ribosomes will stall over the trp codons.
This prevents stem and loop 1 from forming. Eventually stem and loop 2 will be transcribed and form.
This precludes the 3rd stem and loop from formin and thus prevents transcription termination.
RNA polymerase will now progress into the operon allowin it’s expression and the synthesis of more tryptophan.

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84
Q

In 5 bullet point summarise attenuation:

A
  1. Terminator mRNA BEFORE the biosynthetic genes.
  2. It’s formation is prevented if a ribosome stall in a crucial position.
  3. The ribosome is translating a short ORF into a protein that need the AA that is to be synthesised.
  4. So … If AA is present, ribosome won’t stall and terminator will be formed.
  5. Some operon only use attenuation and don’t use repressor sources at all.
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85
Q

What are bacteriapahges?

A

Virus’s that infect bacteria.

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86
Q

What is the structure of the bacteriophage lambda?

A

Has a head and tail structure.

Double stranded linear DNA chromosome of about 48 kilo base pairs.

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87
Q

What are the 4 stages of a hypothetical simple virus?

A
  1. Virus’s DNA (that encodes a single type of viral protein coat) enters the cell
  2. The virus genome is replicated producing multiple copies.
  3. The genome copies are translated and transcribed to produce the viral protein coat.
  4. The viral genomes can the assemble spontaneously with the coat protein to form new virus particles which escape from the cell by lysing it.
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88
Q

How do bacteriophage co-ordinate the expression of genes to facilitate transition through these stages of the developmental program?

A

The first genes to be expressed (usually straight after infection) produce a protein that will allow the switching on of genes that are required later.
These are often referred to as late or early genes.

Early gene expression > protein coded by one early gene > switches on expression of the late genes > late gene expression

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89
Q

What is the choice that the bacteriophage lambda must make? Why must it make this decision?

A

Lysogeny or lytic pathway.

  • if it is going to make lots of new phage particles it is only sensible to make them I’d there are lost of new bacterial hosts for them to invade.
  • therefore if there are not many unoccupied hosts or little nourishment for the virus then it is sensible for them to stay put (the lysogeny pathway).
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90
Q

What is the definition of a temperate phage?

A

It can sit and wait to decide what pathway to choose.
(Phage lambda is an example of this).
A host with a lysogen is immune to further infection.

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91
Q

What are virulent phages?

A

Always opt straight for the aggressive lytic option.

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92
Q

What are bacteriapahges?

A

Virus’s that infect bacteria.

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93
Q

What is the structure of the bacteriophage lambda?

A

Has a head and tail structure.

Double stranded linear DNA chromosome of about 48 kilo base pairs.

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94
Q

What are the 4 stages of a hypothetical simple virus?

A
  1. Virus’s DNA (that encodes a single type of viral protein coat) enters the cell
  2. The virus genome is replicated producing multiple copies.
  3. The genome copies are translated and transcribed to produce the viral protein coat.
  4. The viral genomes can the assemble spontaneously with the coat protein to form new virus particles which escape from the cell by lysing it.
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95
Q

How do bacteriophage co-ordinate the expression of genes to facilitate transition through these stages of the developmental program?

A

The first genes to be expressed (usually straight after infection) produce a protein that will allow the switching on of genes that are required later.
These are often referred to as late or early genes.

Early gene expression > protein coded by one early gene > switches on expression of the late genes > late gene expression

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96
Q

What is the choice that the bacteriophage lambda must make? Why must it make this decision?

A

Lysogeny or lytic pathway.

  • if it is going to make lots of new phage particles it is only sensible to make them I’d there are lost of new bacterial hosts for them to invade.
  • therefore if there are not many unoccupied hosts or little nourishment for the virus then it is sensible for them to stay put (the lysogeny pathway).
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97
Q

What is the definition of a temperate phage?

A

It can sit and wait to decide what pathway to choose.
(Phage lambda is an example of this).
A host with a lysogen is immune to further infection.

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98
Q

What are virulent phages?

A

Always opt straight for the aggressive lytic option.

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99
Q

What happens before phage lambda is required to make the decision between the lititic or lysogeny pathway?

A

The phage attaches to the host cells and injects the lambada DNA. The lambada DNA circulizes

100
Q

Explain when early and late genes are transcribed within the phage lambada.

A
  • early genes must be transcribed as soon as the lambda DNA plasmid has recircularised.
    > these genes direct the synthesis of viral proteins needed for the formation of new virus’s.
    > late genes need to be expressed later prior to assembly of more virus particles and their release from the bacterial cell upon lysis.
101
Q

What is the lysogeny pathway?

A

Phages DNA is integrates into the bacterial chromosome.
Also known and the prophage pathway
Phages DNA is replicated for it by the bacterium
Does NOT result in further infections.

102
Q

What is the lytic pathway?

A

Aim is to create many more phage particles to infect other cells.
Requires synthesis of proteins required to kill the host AND build new phages.
Needs specific genes to be switched on.

103
Q

What enzyme is required in the lysogeny pathway in order to integrate the Phage DNA into the hosts? What is the name given to this process of incorporation of DNA?

A

Intergrase (encoded by an early gene)

- known as conservative site-specific recombination.

104
Q

How does intergrase work to incooperate the phages DNA into the host DNA?

A
  • 2 homologous DNA sequences are present in the host and bacterial DNA.
  • Phage produces the early enzyme intergrase which binds to specific phage sequences on the phage chromosome.
  • the intergrase-phage complex can now bind to the same sequences in the bacterial hosts chromosome.
  • intergrase makes 2 cuts in both chromosomes either side of this region. Intergrase then switches the partner strand and reseals to from a small heteroduplex joint that is 7 nucleotides long.
  • the intergrase enzyme can recognise these new joints as being different from the original sequence in the 2 separate plasmids so can detect whether it needs to act or not.
  • later intergrase is aided by another enzyme, excisase, if the DNA needs to be removed from the bacterial chromosome.
105
Q

What are the 3 stages unique to the lytic cycle?

A
  1. Synthesis of viral proteins needed for formation of new viruses.
  2. Rapid replication of lambda DNA and its packaging into complete viruses.
  3. Cell lysis releases a large number of new viruses.
106
Q

Outline the process regulated by the phage lambda switch.

A
  • developmental program
  • timing of events must be perfect.
    Positive and negative regulation of transcription is used: an absence of activation is not the same as switching off completely.
    > nothing is absolute
  • when we consider the repressor is bound and blocking transcription it just means in most cases or most of the time.
107
Q

What led to the discovery of the phage switch?

A
  • turbid plaques appear when the host is not completely Lysed (I.e. Some lysogeny and some lytic cycle happening.)
  • mutants of phage lambda were identified with clear plaques (only lytic occurring).
  • mutants found to be mutants in one of three proteins names cI/II/III
  • if the phage loses the activity of any of the three proteins it is committed fully to the lytic cycle therefore no lysogeny at all.
    The normal state of affairs is that they will mostly do lysogeny.
  • therefore we can deduce that when functional, each of these three proteteins are either positive factors directing the phage towards lysogeny or factors that repress the lytic pathway.
108
Q

What happens immediately upon circularisation of the phage lambda genome?

A

The bacterial RNA polymerase attaches to two promoters PL and PR to start transcribing the leftwards operon and the right wards operon, ending at the transcription termination signals tL and tR1.

109
Q

Phage lambda: During early transcription what 2 genes are transcribed? And what is its function?

A

N and CRO.

N is an anti-transcription terminator.
- it interacts with RNA polymerase at Pl and PR1 to allow it to pass though the initial terminators and transcribe the left and right operons.

110
Q

Essentially what does the antiterminatior protein N allow?

A

Allows transcription to proceed further. ‘Early transcription’
- it allows polymerase II to transcribe something that is otherwise energetically difficult to trancribe. The action of N allows one signal to be ignored leftwards of the operon and 2 rightwards.

111
Q

After the antiterminatior protein has acted it allows subsequent transcription beyond N (in leftwards) or beyond cro (in the rightwards). What is the difference?

A

Essentially the direction depicts the the operon taken by the phage.
Many of the genes in in the lytic pathway are rightwards of the operon.

112
Q

What is the function of the cro O, P, Q, S and R genes?

A

Cro - is the regulatory gene.
O & P - direct the replication of the phage genome.
Q - switches on the major rightwards operon that expresses all of the head and tail protein genes.
S and R - produce an enzyme that lyse the bacteria from inside.

113
Q

What effect does cro have on the leftwards side of the operon?

A

Cro protein completely inhibits transcription of the leftwards operon.
At high conc it will also reduce expression of the rightward operon.

114
Q

What 3 important proteins are critical to the establishment of lysogeny?

A

Intergrase has always been described for its creation of the prophage.

  • cIII and cII. CII is a very unstable positive control factor (found rightwards).
115
Q

Explain the generation of the lambda repressor.

A

CI gene encodes the lambda repressor, and sits between the left and right promoters.

116
Q

What is the function of the lambda repressor?

A

Represses the lytic cycle and allows establishment of lysogeny.

The lambda repressor gene (cl) is not under the control of either the leftwards or rightwards promoter so it must have its own promoter somewhere else in order to be able to function.

117
Q

How is the expression of c1 started- hence the establishment of lysogeny?

A

CII is stabilised by the CIII protein.

  • CIII acts as an inhibitor of the bacterial host’s own pro teases that destabilise cII.
  • there are two proteases; HflA and HflB that both destabilise CII.
  • if these are inactivated the cII is more stable. In addition expression of the HflA and HflB proteins is subject to catabolise repression.
  • Sugar starvation leads to higher frequency of lysogeny is dictated by the growth conditions of bacteria.
  • so all in all the establishment of lysogeny is dictated by the conditions of the bacteria.
118
Q

What is the function of CII?

A
  • CII stimulates the transcription from the PRE promoter.
  • this promoter for repressor establishment.
  • this allows the expression of the c1 gene which encodes the lambda repressor.
119
Q

What is the function of the lambda repressor?

A

It inhibits expression of the left and right operons including CII and CIII.

120
Q

If the lambda repressor prevents further production of CII and CIII which are used to produce CI how are levels of lambda repressor protein kept high enough to maintain lysogeny?

A

We require another promoter PRM to continue transcribing CI.

121
Q

How is lysogeny maintained?

A

Only CI is being transcribed as both PL and PR are now blocked by the lambda repressor.
CII and CIII got CI expression started but they cannot continue the job as they are both on operons that are no longer expressed.
- now lambda repressor has to take care of its own expression; it positively regulates its own transcription.

122
Q

What are the functions of the following genes in the lytic pathway? Cro, O&P, Q, S & R?

A

Cro = regulatory protein
O & P = initiate phage replication.
Q = positive control factor. Switches on major rightwards operon (it’s another antiterminatior protein)
S & R = endolysin.- like lysozyme digest back ethical cell walls.

123
Q

Explain the interaction of CRO and CI.

A 
Lambada repressor (CI) inhibits leftward AND rightwards but promotes itself. 
CRO blocks binding of lambda repressor at these 2 promoters. 
We need to remember the promoters have operator regions.
124
Q

What effect does the CRO protein have on the operator region?

A

It blocks the operator region for PR and PL.

125
Q

How are CRO and CI in conflict?

A

When CRO is repressed it also wants to occupy the operator regions at the right and left promoters, like lambda.

  • however it does so with different affinities.
  • this turns out to be important later, lambada aim is to cause lysogeny pathway to be adopted; CRO’s aim is to promote lytic pathway.
126
Q

What is the decision between lysogeny or lytic pathway decided by?

A

The binding competition of CRO and CI (repressor) protein a the rightwards of the operator.

127
Q

Explain where and what happens when CI binds.

A

CI binds first to OR1 (because it’s affinity is highest for this part of the operator region. And because this region overlaps with the rightwards promoter (PR), this inhibits the transcription of the rightwards operon (all of the lytic genes AND CRO)

128
Q

What happens when OR2 is bound by CI proteins?

What is this regulation known as?

A

It inhibits further transcription from PRM (as it is blocked and RNA pol cannot access PRM). Thus CI protein regulates its own expression.
- autogenous control.

129
Q

How is the bind of CRO to the rightwards operator different to CI?

A

CRO proteins binds in reverse order as binding affinity for the different ORs is different.

130
Q

Explain the mechanism of how CRO binding is different to CI.

A
  • first it binds to OR3 which prevents CI expression (because it blocks PRM) then OR2, which prevents CI protein from stimulating its own expression (because CI cannot get there). And lastly it bind to OR1 which reduces the expression of the rightwards operon.
  • eventually high levels of CI inhibits its own transcription.
131
Q

Which protein normally wins the battle and why?

A

CI nearly always wins if it is there, so to get the alternative pathway to happen it needs to be removed. The decision is made by earl/late expression.

132
Q

What is breaking lysogeny?

A

Waking up the system so that it can change from the dormant lysogeny state to the lytic state. For this to happen we need to stop CI inhibiting both directions.

133
Q

How can lysogeny be broken?

A

We need to xis (excise) to release the phage DNA from the lysogen and we need the rightward operon to be expressed so we get all the proteins needed for phage coat protein, lysis etc.

134
Q

What happens if we get rid of the CI repressor?

A

We get out of lysogeny.

135
Q

What gene is usually used to repress the SOS response in E.cloi?

A

LexA gene

136
Q

How does the LexA gene effect the lambada repressor?

A

Damaged DNA binds to RecA protein (involved in repair process) and activates its protease function. RecA protein cleaves LexA protease function. RecA protein cleaves LexA protein to activate SOS response.
- as a by product it can also cleave the lambda repressor (CI).

137
Q

Explain the regulation of RecA.

A

RecA is a DNA repair protein needed when the cell’s DNA is being damaged (e.g. By UV)

  • when DNA is damaged, single stranded DNA molecules bind to RecA and activate its protease activity.
  • RecA can now cleave LexA and CI.
138
Q

What happens once CI protein is inactivated?

A
  • transcription can start from PL and PR again.
  • Excisase and intergrase proteins are made to remove the prophage from the bacterial genome, which now enters the lytic pathway.
139
Q

How are attempts of CIII/CII proteins to make more CI doomed?

A
  • Rec A will inactivated it.
140
Q

What is used in translation?

A
  • ribosomes
  • tRNA
  • initiation factors
  • elongation factors
  • releasing factors
  • amino acids
  • amino yo synthetases
141
Q

Explain the composition of ribosomes.

A

EUK and PRO both = large and small subunits.

  • both are made of RNA and protein, with many different proteins per subunit.
  • in terms of secondary structure the RNA predominates. The large subunit is made up of more than one RNA molecule; in bacteria these are the 23S and 5S.
  • there is a small subunit too counting a single RNA (bacteria= 16S and euks 18S) . There are many proteins incoperated also.
142
Q

How are ribosomes measured?

A
  • increasing sucrose gradient in tube.
  • cell extract on top of gradient
  • centrifuge 500,000 x g for several hours
  • cell components form bindings dependant on their sedimentation coefficients.

> the rate depends upon side, density and shape- hence its non-linear character.
Svedberg or S units are non-linear. They measure the rate at which a particle sediments of a liquid when subject to high centrifugal force.

143
Q

What is the active site of a ribosome called?

A

Peptidyl transferase.

  • n.b. For many years this enzyme activity was though to be a property of one of the proteins comprising the ribosome, but now we know that peptidyl transferase activity is conferred by one of the rRNAs.
144
Q

What is the importance of the stable secondary structure forming the small subunit?

A

SO that the accessory proteins can attach to a scaffold.

- the secondary structure is crucial as it is the rRNAs that actually catalyse the peptide bond.

145
Q

AA are selected for the growin polypeptide chain in the order specified by mRNA’s but what brings them?

A

transfer RNA specific for that amino acid.

146
Q

Describe the structure of tRNA and how it relates to its function.

A

Often described as having a clover leaf structure.
There are 2 crucial regions.
- the anticodon
- the acceptor arm

In this diagram R stands for purine base, Y for pyrimidine and (funky y) pseudoridine.

147
Q

What does the anticodon on tRNA do?

A

It is complementary to the codon in mRNA, it is the bit that reads the code.

  • the 3’ end always ends in CCA.
  • the terminal adenine is the one that is covelently linked to the specific amino acid.
148
Q

What causes specificity? (Anticodon)

A

Specificity results from the shape and modifications to tRNA, this allows tRNA to be recognises by its specific aminoacyl tRNA synthetase.

149
Q

What is the job of the receptor arm? HOw is it formed?

A

Binds to an amino acid.

- this arm is formed by the binding together of 7bp of the 5’ and 3’ ends of the molecule. AA attaches to the 3’ end.

150
Q

What is the d-loop?

What is the T(cool y)C arm?

A

Has modified Dihydouridine base.
Names after the sequence thymidine-pseudoridine-cytosine.
- pseudoridine is another modified uridine base.

151
Q

What is the key enzyme involved in tRNA protein synthesis?

A

Aminoacyl tRNA synthetases are they key enzymes. Each one recognises the correct amino acid to link to the correct tRNA.
There is one aminoacyl tRNA synthase for each of the twenty amino acids.

152
Q

Explain the role of Aminoacyl tRNA synthetase join the amino acid to its tRNA.

A
  • 1 enzyme for each amino acid (20)
  • Hihgly specific
  • activate amino acid with ATP
  • energy maintained when linked to tRNA
  • Energy for protein synthesis comes from this chemical bond.
  • AA is now a passenger to the tRNA.
153
Q

What are the group of enzymes responsible for catalysing the 2-step reaction which is Hihgly specific; there is 1 enzyme for each amino acid?

A

Aminoacyl tRNA synthetases.

154
Q

What is the first step of tRNA synthesis?

A

An activated amino acid intermediate is produced that has a link between its carboxyl group and AMP.

  • in this reaction ATP is cleaved and this produces energy to join the amino acid to the tRNA.
  • the amino acid-AMP remains bound to the enzyme whilst the second stage occurs.
155
Q

What is step 2 of amino acid synthesis

A

Here the tRNA takes the place of the AMP so that aminoacyl-tRNA can form.

  • Amino acid is now a passenger to the tRNA.
  • in this way each tRNA forms a covalent linkage with its specific amino acid by aminoacylation (also known as charging).
  • the amino acid becomes attached to the acceptor arm, linked by a go vent linkage between the AA- COOH group and the 2 or 3’ OH group on the terminal nucleotide of the tRNA (which is always A).
156
Q

What contributes to the accuracy of amino acid binding?

A

The two stage action and the editing site on the amino acid tRNA synthetase.
- tRNA synthetases remove their own coupling errors though hydro lytic editing of incorrectly attached amino acids. The incorrect amino acid is rejected by the editing site.

157
Q

What are the three steps of translation?

A

Initiation
Elongation
Termination

158
Q

What is always the first amino acid in translation?

A

Always methionine.

In bacteria the first methionine is modified by the addition of a formal group.

159
Q

What is the name given to the tRNA that brings the first AA the methionine?

A

Brought to the ribosome by the initiator tRNA.
> tRNAAfMet
- In bacteria ONLY this initiation methionine has a formal (-HCO) group attached to the N of the amino group hence N-formal methionine.

160
Q

Why do proteins not always have an N terminal methionine?

A

Many proteins are extensively modified after translation and the methionine are lost.

161
Q

What are the initiator tRNAs in bacteria and eukaryotes? (Hint: they’re different)

A

Bacteria use - Met-tRNAFmet

Eukaryotes use - Met-tRNAimet

162
Q

What is initiation step 1?

A

Assembly of the small subunit

163
Q

In bacteria what is the translation initiation complex?

A
  • it is built up directly at the initiation codon, where protein synthesis will begin.
  • the large and small subunits remain separate in the cytoplasm until they need to assemble.
164
Q

In bacteria when does the process of translation initiate?

A

When the ribosome assembles on the mRNA at a special ribosome binding site.
- it can find where to bind in bacteria because there is a sequence upstream of the first codon (AUG) that is complimentary to a short sequence of 16s rRNA.

165
Q

What is the name given to the 16srRNA sequence?

A

Shine-Dalgarno sequence.

166
Q

What do eukaryots have instead of the Shine-dalgarno sequence?

A

Eukaryotes recognise the 5’-CAP.

167
Q

What is the function of the small subunits accompaniment by IF-3?

A

It prevents the large subunit from attaching until the appropriate time.

168
Q

What is the function of the IF-2 complex?

A

It brings the initiator tRNA to the right position along with a molecule of GTP.

169
Q

How is the initiation of translation completed?

A

By attachment of large subunit.

IF-1 is also involved at this stage and AIDS binding of the large subunit (50s) and ensures correct assembly of 30s and 50S.

170
Q

What process facilitates the attachment of the large subunit?

A

It requires energy and this is obtained by IF2 hydrolysing it’s bound GTP to GDP, and results in release of the initiation factors.
- this makes the process irreversible.

171
Q

Once the assembly of the small (30s) and large (50s) subunits is complete …

A

This constitutes the assembly of the whole 70s complex.

172
Q

What are the names of the 3 sites on the ribosomes for binding tRNA moleucles?

A 
A = Aminpacyl-tRNA site
P = Peptidyll- tRNA site
E = Exit site
173
Q

What happens at the A site of the ribosome?

A

The tRNA must be bound to an amino acid that corresponds to the AA dictated by the mRN currently in the A site.

174
Q

What happens at the P site of the ribosome?

A

This holds a tRNA bound to the growing polypeptide chain.

175
Q

What happens at the E site?

A

This releases the now empty tRNA from the ribosome.

176
Q

What is special about Met-tRNAfMEt?

A

It is the only aminoacyl-tRNA that can load directly into the p-site.

177
Q

What happens in phase 2 (elongation)?

A

Similar in bacteria and eukaryotes

  • individual amino acids are attached to the carboxyl (COOH) terminus of the growin polypeptide- the first peptide bond.
  • Assembling the ribosome out of the 2 subunits has created 2 sites at which aminoacyl tRNAs can bind.
178
Q

Elongation requires 3 elongation factors what are they, and what are their functions? (In bacteria)

A

EF1A
> loads AA-tRNA into the A-site. Commonest protein in cell.
EF-1B
> recycles EF-Tu
EF-2 (EF-G)
> checks that translocation is successful.

179
Q

What does EF-1A do in order to try and progress elongation?

A
  • It complexes with GTP and aminoacyl-tRNAs.
  • there are multiple complexes in the cell, each trying to load their AA into an empty A-site.
  • only one will succeed, this is dictated by the codon in the mRNA.
  • mischarged tRNAs will be rejected.
  • the codon and anticodon must match.
180
Q

Once the correct AA-tRNA is loaded into A site what happens?

A

EF1-A will hydrolyse its GTP to release energy allowing the formation of a peptide bond by the ribosome peptidyl transferase is an irreversible process.

181
Q

How does EF-1B recycle EF-1A?

A

By removing the GDP and allowing free GTP to bind.

EF-1A can one pick up a fresh AA-tRNA.

182
Q

What is translocation?

A

The first 2 AA have been joined, the attachment between the first amino acid and its aminoacyl tRNA are broken.

  • translocation is the process in which the ribosome moves 3 nucleotides along the mRNA, ejecting the deacylated initiator tRNA from the P-site, which now becomes occupied by the second tRNA attached to the first 2 amino acids of the polypeptide.
  • a new codon can now enter the A site.
183
Q

What does translocation require?

A

Hydrolysis of a molecule of GTP and is mediated by EF-2. In bacteria (not euk) the ejected aminoacyl tRNA leaves via the third site the E site.

184
Q

Where does the synthesis of the peptide bond occur in the ribosome?

A

Occurs in the p-site.

185
Q

What is the enzymatic function is peptidyl transferase catalysed by?

A

The 23S rRNA in the large subunit.

186
Q

What is the peptide bond synthesised by?

A

The transfer of the growing polypeptide chain from the p-site tRNA to the free amino group on the amino acid on the tRNA in the A-site.

187
Q

What occurs during termination of translation?

A
  • requires a stop codon

- RF1 and RF2 are proteins that mimic AA-tRNA and can load onto the A site.

188
Q

What do RF-1/2/3 do?

A 
RF-1 = recognises UAG and UAA
RF-2 = recognises UGA and UAA
RF-3 = stimulates dissociation of RF1 and RF-2.
189
Q

What happens when a terminator codon moves into the A-site?

A

It acts as a signal for the ribosomes to release the now complete protein

190
Q

How are RF1/2 proteins capable of loading into the A-site?

What happens once they get their?

A

They mimic AA-tRNA.

The bond between the last amino acid of the protein and the adenine of the tRNA in the P-site is hydrolysed, releasing the protein.

191
Q

What is the function of the ribosome recycling factor (RRF) ?

A

The ribosome is separated into its two subunits.

192
Q

List 2 key features of the genetic code.

A
  • the code is universal

- the code is degenerate

193
Q

How does altering the 1st amino acid of a codon compare to altering the 3rd?

A

Altering the first base will change the amino acid considerably.
Altering the 3 will usually have no effect.

194
Q

What is the term ‘wobble’ referring to?

A

Term used to decibe the fact that on tRNA can recognise more than one codon for the same amino acid if the codon differs only in its third position.

195
Q

List 7 important things associated with eukaryotic transcription.

A
  1. Separation of transcription and translation
  2. More than 1 RNA polymerase
  3. Transcription factors
  4. Extra proteins associate with holoenzyme.
  5. The mediator complex
  6. Transcript processing
  7. Hi stones regulate access to promoters
196
Q

Explain what is meant by separation of transcription and translation?

A
  • mRNA is exported from the nucleus.
197
Q

Where is RNA POL I found what does it synthesise and what is its alpha-amanitin?

A

Found: nucleoli so
Synthesises: rRNA
Alpha- amanitin sensitivity: none

198
Q

Where is RNA polymerase II found, what does it synthesise and what is its alpha-amanitin sensitivity?

A

Found: nucleoplasm
Synthesises: hnRNA (protein coding genes)
Alpha- amanitin sensitivity: high

199
Q

Where is RNA polymerase III found, what does it synthesise and what is its alpha-amantin sensitivity?

A

Found: nucleoplasm
Synthesises: tRNA, other small nuclear RNAs (snRNA)
Alpha- amanitin sensitivity: medium

200
Q

What are the 2 other polymerises found in plants and what do they produce?

A

RNA pol IV and V both involved in the production of small interfering RNAs (siRNAs). These are negative regualtory moleucles that reduce levels of transcription.

201
Q

What is special about mitochondrial and chloroplasts?

A

They have their own RNA polymerase to transcribe just their own genomes.

202
Q

Read info about discovery of eukaryotic polymerises.

A

Jbfdiof

203
Q

How do prokaryotes and eureka ores differ in terms of transcritpition factors?

A

Eukaryotes uses positive factors (activators) more than repressor a and transcription cannot proceed without activators. This differs from prokaryotes because for them transcription CA N proceed provided RNA polymerase is present, in the right place and unimpeded.

204
Q

What are GRPs?

A

Gene regulatory proteins. In other words transcritpition factors.

205
Q

What part of the promoter do transcription factors bind?

A

They bind to recognition sequences called promoter motifs or cigs-acting elements.

  • unlike in bacteria these can be far away from the gene in question yet still influence its expression.
  • when the transcription factor and cis-element come together RNA Pol II can start working.
206
Q

what do GTFs do?

A

They are initiation factors, they position Pol II in the right place ready for transcription, and they help to separate the strands so the template can get to the active site of the enzyme.

207
Q

What does TFIID = ?

A

TBP (TATA box binding proteins) + 13 TAFs (TBP associated factors)

208
Q

What does the pre initiation complex refer to?

What happens when it is assembled?

A

Pol II + GFTs

Transcription is ready to begin.

209
Q

What did Young and Kornberg do and what did they discover by doing so?

A

In a lab they added TFs (activators), Pol II and GFTs to promoters in a cell free (in vitro) and transcription did NOT occur as they expected.
- they concluded that a co-activator was required.
The mediator complex was discovered.

210
Q

What is the structure of the mediator complex (in yeast but assumed to be the same in other organisms)?

A

In yeast the tail binds TFs, head binds Pol II.

- The kinase domain regulates the function of the whole complex.

211
Q

How does the mediator complex work?

A

Loop the DNA allows mediator to bring transcription factors and their cis elements into close promixmity with Pol II, now Pol II can transcribe the gene.

212
Q

What is transcript processing?

A
  • mRNA is exported from the nucleus.
213
Q

Upon basic inspection what is different about eukaryotic and prokaryotic transcription?

A
  • In bacteria each coding sequence within this mRNA is translated quite seporately. Such mRNA is known as polycistronic mRNA. Eukaryotes cannot do this, as their mRNA is quite different.
  • eukaryotic DNA only contains one coding sequence, which is often interrupted by introns. These are removed by splicing. After transcription there are further modifications to the mRNA.
    > a 5’ CAP is added and a polyA-tail. Introns need to be spliced out. Polymerase II synthesises RNA whilst co-ordinating processing at the same time so it needs to have all these functions.
214
Q

What is a 5’ CAP?

A

The 5’ CAP I’d throw addition of a GTP molecule to the 5’ end of the mRNA. The GTP is further modified by the addition of a methyl group.
- this helps to stabilise the mRNA

215
Q

How is the 5’ CAP added?

A

It is added by proteins that interact with the CTD domain 9the C-terminal domain of RNA polymerase).

216
Q

What does the CAP prevent?

A

RNA degradation.

It is also an identifier that shows this molecule is an mRNA.

217
Q

Explain the placing of the 5’ CAP

A

The CAP can only be added to a diphosphate or triphosphate group.
- so if a moleucle of mRNA is broken in the middle, only a mono phosphate would be present at the 5’ end, this wouldn’t get capped and so the RNA molecule wouldn’t recognise it as a transcritpition.

218
Q

How is a PolyA tail added and what is its purpose?

A

200-250 A residues are added to the 3’ end of the mRNA by the Enzyme PolyA-polymerase.
- these additions help to stabilise the mRNA.

219
Q

What is AAUUAA a type of sequence of? What does it do?

A

Signals the point where the mRNA must be cut off. After this the A residues are added.

220
Q

How does splicing of transcripts occur?

A

Occurs in the nucleus with the aid of many snRNA moleucles and their associated proteins.

221
Q

How do snRNAs work?

A

They help recognise exactly the exon-intron boundaries. A specific adenine attacks the boundary on the 5’ side of the intron and causes cleavage so that there is now a free intron end to which the adenine can become covalently bound.

  • A further snRNA initiates the formation of the lariat and promotes the intron excision. This is an exact and complex process.
  • Introns are not removed in a particular order I.e. The 1st intron, then the 2nd intron and so on.
222
Q

Read the bit below this section heather 4/4

A

Don’t really understand it otherwise.

223
Q

How can histone modifications control whether the DNA can be transcribed?

A

Nucleosomes are comprised of 4 types of histones (2 of each).
- histones are linked to essentially every cellular process requiring DNA access, including transcription.
- there are a number of enzymes that add or remove chemical groups onto or from the N terminal protein tails of the histones and regulate the histones ability to bind the DNA tightly or not.
> thus regulating histone tails’ modification is a way of regulating access of the DNA by Pol II.

224
Q

How can histones be modified?

A
  • methylation can occur to lysine or arginine in several ways.
  • phosphorylation
  • acetylation
  • ubiquitination

Most modification shown add a relatively small molecule onto the histone tails. With the exception of ubiquitination.

225
Q

What does acetylation target, and what does this modification do?

A

Targets lys residues in the amino-terminal tails of the core histone proteins.
Acetylation of the tail domains inhibits the folding of Nucleosomes arrays into secondary and tertiary chromatin structures.
- modification of H2B and H4 have the greatest effect on tertiary structure formation.

> thus histone tail acetylation results in chromatin decondensation, thereby allowing access to transcription factors and other transcription co-activators.

226
Q

What are eukaryotic genes switched on or off in response to?

A

Diverse signals.

227
Q

Why may genes only be expressed in one cell type and not others?

A

Each cell has a different role in the whole organism so it doesn’t necessarily need to produce the same proteins, so some genes can be switched off.

228
Q

What are three reasons for measuring transcript levels?

A
  1. It is a way of detecting what and organism has responded to and a way of seeing what the response is.
    - a developmental signal
    - an external condition.
  2. It is a way of diagnosing the effects of a genetic difference.
    - in humans it might mean looking to see if a patient can express a particular gene.
  3. It’s also a way of predicting which proteins might be produced.
    - changes in transcript levels usually precede changes in protein levels- also chapter and easier to measure.
229
Q

How can we measure transcript levels? (These methods refer to eukaryotes)

A 
A) hybridisation- northern blots
B) cDNA- based methods.
1. QRT-PCR
2. microarrays
3. Next generation sequencing.
230
Q

What are the 4 stages involved in producing a northern blot?

A
  1. First extract total RNA from the cells. Make a radioactively labelled ssDNA probe. You choose the sequence of this probe to match the mRNA you want to detect.
  2. You transfer the RNA from the gel onto a nitrocellulose filter by blotting with a weight.
  3. Once the RNA has be transferred to the filter, the filter is mixed with radioactive probe and allowed to hybridise. During this mRNA molecules on the filter that bind and recognise the radioactive probe will do so. MRNA for every other gene that is not a match will not bind any radio active. When enough time has elapsed for hybridisation, uninoperated probe is washed away, leaving only the probe that is attached to the mRNA molecules on the filter.
  4. X-ray film is used to detect where the highest levels of radioactive are on the filter. Dark band show samples that have a high level of radioactivity and therefore must have a high level of transcript. In the blot shown on the slide the very dark bands show high levels of expression of the desired gene.
231
Q

How does the northern blot work?

A

Capillary action of the paper draws buffer through the stack drawin RNA onto the filter.

  • filter is incubated with radioactive DNA probe for the gene of interest.
  • all of the many RNAs present in your sample will be spread out on the gel.
  • if you transfer all of the mRNA bands from the gel to a more solid matrix called a filter or membrane.
  • transfer of the RNA to the filter happens by capillary action. Liquid buffer is drawn through a pile of filter papers and the tissue and this draws the RNA though the gel and onto the filter where it can later be immobilised so it doesn’t come off again.
  • once all the RNA molecules are attached to the filter you only want to display the ones you’re interested in.
  • so you label up just your transcript of interest by incubating the membrane with radioactive DNA probe corresponding to the sequence of interest.
232
Q

What is the idea behind cDNA based methods?

A
  • non-radioactive
  • can measure the transcripts from many different genes in one experiment
  • most use amplification methods so need only small amounts of material
  • BUT cannot do PCR on RNA
  • we must convert RNA to DNA
233
Q

How can we convert RNA to DNA?

A

Using the enzyme reverse transcriptase.

  • it opposes the central dogma.
  • comes from virus with RNA genome that need to make DNA to infect host.
234
Q

How is DNA made from RNA?

A

Always need a primer.

  • every mRNA will have a poly A tail so the same primer can be used throughout; poly T primer.
  • once it binds reverse transcriptase (RT) extends the nucleotide chain from the primer back towards the 5’ end of the mRNA and makes a copy.
  • Now we have 2 strands one DNA and one RNA we don’t need both so the RNA is degraded by RNAse
235
Q

How can we make our single stranded DNA double?

A

We must synthesise a complimentary DNA strand a DNA polymerase can do this.

236
Q

Explain the logic behind ‘ more mRNA = more cDNA copies)

A

If your sample has a high level of transcription you will have a lot more mRNA.
> this will lead to the formation of a lot of cDNA.
Therefore cDNA levels are proportional to mRNA levels and therefore can be used as a proxy for measuring RNA.
CDNA is a lot easier tot handle than RNA as it doesn’t degrade so easily.

237
Q

What is the other advantage of making cDNA?

A
  • you can do PCR on cDNA so now we have an amplification method.
238
Q

What are the 3 steps of PCR?

A
  1. Heat to separate strands
  2. Cool to anneal primers
  3. DNA synthesis.
239
Q

What happens if you do PCR using primers of the transcripts we want to study?

A

Although cDNA contains a copy of EVERY transcript in your sample, you only amplify the one transcript you want to know about.

240
Q

How can we quantitatively measure the quantity of product? What is the problem with using a gel?

A
  • when using a gel we tend to see the end result where the 2 samples look very much alike, because the slow one catches up worth the faster one.
  • if we look at and analyse the reaction when in the exponential phase we will be able to see this.
241
Q

What is the advantage of fluorescence over a gel for detection of PCR product?

A
  • a flour net dye binds to newly synthesised DNA PCR product.
  • fluorescence gets BRIGHTER as the amount of product increases.
  • amount of product can be monitored in REAL TIME throughout the reaction.
  • called qRT-PCR
242
Q

Once the flour sense is done, what’s next how do we use it?

A

96-well plate is used.
- an excitation light source excites the fluorescent dye bound to the PCR product and then a picture of the resultant light is emitted.
PMTs are photomultiplier tubes- electronic detectors that sense and amplify light signals including fluorescence.

243
Q

How can 2 samples be compared?

A

MRNA is extracted and labelled with either a red or green fluorescent dye, then labelled cDNAs are mixed and allowed to hybridise to the microchip arrays.
- the probes on the chip for every gene will bind red or green copies of the cDNA for that particular gene. Then the red and green signals are scanned by a laser and recorded.

> each spot represent one gene

  • each spot can bind to the corresponding red and green labelled cDNA
  • amounts of cDNA relate to number of copies of corespondent transcript.
  • increasing fluorescence intensity means more cDNA copies of the gene = more transcripts.
  • the more cDNA copies are present to bind to the probe, the stronger the intensity of the red or green colour.
244
Q

What happens to compare the intensity of red or green signals?

A

They are scanned to give a ration of expression.

245
Q

What is next generation sequencing?

A

RNAseq can report expression of EVERY gene.

  • next generation sequencing (NGS) determines sequence of millions of DNA molecules.
  • convert mRNA to cDNA
  • sequence all the copies

> it is a new technique that allows millions of DNA sequences to be ‘read’ in short space of time.
- it can be used to sequence entire genomes but it is also useful for measuring levels of mRNAs. Usually convert RNA to cDNA first.

246
Q

How is NGS interpreted?

A

If you sequence the same bit of cDNA lots of times it means you have lots of mRNA.

247
Q

Compare advantages of microarrays and RNAseq

A

MICROARRAYS

  • requires a chip with a probe for every gene. Need to know the genome sequence.
  • Data is fairly simple to analyse (ratio btw green and red)
  • family expensive

RNAseq

  • less genome information required. Good for less well studied genomes.
  • RNAseq DNA is complicated to analyse.
  • Fairly expensive.

Genetics (5 decks)

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