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BMS 242 - Advanced Cell and Molecular > Control of gene expression: DNA > Flashcards

Control of gene expression: DNA Flashcards

1
Q

How are differences between cells brought about when they all contain the same genes?

A

Differential gene expression - only a fraction of the genes in the cell are expressed

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2
Q

What is the most important change in a cell in a diseased state?

A

Expresses different set of genes

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3
Q

What causes cells to change gene expression?

A

In response to signals and other cues in the environment

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4
Q

What regulates the level of expression of a gene in the cell?

A

The level of the transcript, regulated by transcription factors

(Lots of RNA –> lots of proteins)

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5
Q

What charge do DNA binding proteins have?

A

Positive (as DNA is negatively charged)

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6
Q

Where do DNA biding proteins reach into on DNA?

A

The major groove

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7
Q

What are the 3 positively charge amino acids which help DNA binding proteins to be attracted to DNA?

A
  • Arginine (Arg)
  • Leucine (Leu)
  • Histadine (His)
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8
Q

Where do GENERAL transcription factors bind to DNA?

A

Response element - short stretch of DNA within a promoter

Cis

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9
Q

Out of DNA and proteins, which has a limited topology?

A

DNA

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10
Q

How do GENERAL transcription factors recognise response elements in promoter?

A
  • Proteins reach into the major groove and interact with groups which decorate the outside of the double helix, is a specific pattern
  • H-bond acceptors and H-bond donors, as well as methyl groups (hydrophobic)

ALSO

  • Amino acid side chains from a-helice/b-sheet on the TF dangle into the major groove and make contacts with he DNA
  • Can form H bonds with BASES
  • Binds to many places on the DNA to hold the transcription factors down
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11
Q

What group in DNA is hydrophobic?

A

Methyl group CH3

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12
Q

What is needed to stabilise the transcription factor on the DNA?

A

Multiple connections between the bases on DNA and the amino acids on the transcription factor

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13
Q

What is Rox1 and where does it bind?

A

Rox1 is a transcription factor from yeast

Binds to 8 DNA sites in 3 different yeast GENES

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14
Q

Where are the binding sites for Rox1?

A

In yeast:

3 sites in HEM13 gene
4 sites in ANB1 gene
1 sites in ROX1 gene

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15
Q

Why does Rox1 transcription factor bind to its own gene?

A

So it can regulate itself

Negative feedback loop

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16
Q

What is the structure of the Rox1 binding site in genes?

A

All are different but all contain GTT in the centre of the site

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17
Q

What are 3 ways to compare the binding sequences of a transcription factor?

A

1) Compare the frequency at each position
2) Make a consensus sequence
3) Make a sequence logo

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18
Q

What is a consensus sequence?

A

A way of comparing the biding sequence of sites for a specific transcription factor:

  • Write a sequence with the most COMMON base at each position
  • Where unsure - put a different letter
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19
Q

What is a sequence logo?

A

A way of comparing the biding sequence of sites for a specific transcription factor:

  • Draw the letters of the bases different sizes, depending on how common they are at that position
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20
Q

What can a consensus sequence be used for? (2 things)

A

1) To identify more genes regulated by the transcription factor
2) Identification of the sequence in other organisms and cells - homologues

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21
Q

Describe the affinity of the DNA biding sites for a transcription factor

A

Have DIFFERENT affinities, but when the transcription factor increases to a certain level - all become saturated

Site with the highest affinity is the FIRST one that transcription factor binds to - will also be the site which is occupied most of the time

However, binding is TRANSIENT

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22
Q

Why is the binding between a transcription factor and the binding site on DNA not perfect?

A

Ideal to have TRANSIENT binding to be able to MODULATE the levels of gene expression within the cells

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23
Q

In the DNA sequence, what is conserved between closely related species?

A
  • Coding sequence (exons)

- Regulatory sequences in non-coding regions (introns)

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24
Q

What did alignment of the genes in closely related species identify?

A

Conserved sequences in introns, which are important regulatory sequences

Proteins which bind to these sequences

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25
Q

What are motifs which allow proteins (such as transcription factors, activators and repressors) to BIND TO DNA?

(4 of them)

A

1) Helix-TURN-helix
2) Zinc fingers
3) Leucine zippers
4) Helix-LOOP-helix

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26
Q

What is the structure of a helix-TURN-helix?

A

2 helices:
- RECOGNITON helix - inserts into the major groove and makes specific contacts with the DNA with its amino acids

  • Other - stabilises the position on DNA
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27
Q

How does a helix-TURN-helix motif bind to DNA?

A

As DIMERS to 2 consecutive major grooves on DNA

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28
Q

What is the structure of the DNA binding sequence which helix-TURN-helix bind to?

A

Palindromic sequence

AACAC

29
Q

Why do transcription factors have multiple binding motifs?

A

To bind to DNA in many places

30
Q

How does a zinc finger domain interact with DNA?

A

Alpha helice - recognises 2 bases in the major groove

Wraps around the DNA

Uses arginine and histine to interact with the bases on DNA (form H bonds)

31
Q

What is the structure of leucine zippers?

A

Alpha helical monomers held together by hydrophobic amino acids to make a DIMER

Dimer forms a ‘clothes peg’ shape and interacts with 2 positions in the major groove

32
Q

How can leucine zippers dimerise?

What does each type bind?

A

With themselves - homodimers
(Can bind symmetrical sequences)

With different leucine zippers - heterodimers
(Can bind to non-identical sequences)

33
Q

What is the helix-LOOP-helix a modification of and how?

A

A leucine zipper

Has a loop in the structure

34
Q

What does the helix-LOOP-helix structure allow?

A

Flexibility in how they orient in DNA

35
Q

How can helix-LOOP-helix dimerise?

A

Homodimers

OR

Heterodimers

36
Q

Why do many transcription factors bind as dimers?

A

Co-operativity

Dramatically increases binding strength - synergy (combine to produce an affect greater than the sum of their separate effects)

37
Q

What is the structure of a transcription factor? (what modules (4))

A

Modular structure:

1) DNA binding domain
2) Protein binding domain
3) Regulatory domain
4) Activation domain

38
Q

What does the protein binding domain of a transcription factor allow?

A

Dimerisation of the transcription factors

39
Q

What does the activation domain of a transcription factor allow?

A

Interaction with the TRANSCRIPTION INITIATION COMPEX (RNA pol II)

40
Q

What can be used to identify transcription factors which bind to a specific stretch of DNA?

What do both of these methods use?

A

EMSA (electrophoretic mobility shift assay)

DNAse I footprinting

BOTH involve gel electrophoresis

41
Q

What is the process of EMSA?

A

1) Start with a sequence which contains the regulatory region (binding site)
2) Radioactively label one end of the DNA using 32P
3) Mix the DNA with cell extract (or purified transcription factor) and incubate
4) Run samples by gel electrophoresis
5) Expose the gel to identify radioactively labelled DNA

42
Q

In ESMA, what cell extract is the radioactively labelled DNA mixed with?

A

Purified proteins from the cells which are thought to regulate the gene (where the gene is expressed)

43
Q

In gel electrophoresis, what molecules travel faster in the gel?

A

Smaller molecules

44
Q

In ESMA, when exposing the gel to show the radioactively labelled DNA, what pattern is discovered and why?

A
  • Most of the radioactively labelled DNA is located at the bottom of the gel (no TF bound, smaller, migrates through the gel quicker)
  • In some samples, there is a band of radioactivity further up on the gel (migrated slower)
  • Suggests have transcription factor bound which retards migration through the gel
45
Q

What is the process of DNAse I footprinting?

A

1) Radioactively label one end of DNA using 32P
2) Mix with cell extract (or purified transcription factor) - some proteins will bind to the DNA
3) Add DNase in very low concentrations - partially digest the DNA
4) Run the sample on a gel using gel electrophoresis- identify what TF binds to the DNA sequence

46
Q

What is a probe?

A

Radioactively labelled portion of DNA

47
Q

What does the gel look like after running gel electrophoresis in DNAse I footprinting?

A

Ladder-like effect where the position on the ladder represents the size of the sample

48
Q

In DNAase I footprinting, what happens if the cell extract is not added and why?

A

Bands of MANY sizes, all the way through the gel - TF doesn’t protect the DNA binding site from being cut

If cut close to the probe - small fragment at the bottom of the gel

If cut far from the probe - long fragment near the top of the gel

49
Q

In DNAase I footprinting, what happens if the cell extract added contains a TF which binds to the DNA biding site and why?

A

Window where there is no bands of a certain size formed, due to physical blockage of the DNAse to these sites by the transcription factor

50
Q

In DNAse I footprinting, what does heat do?

A

Destroys the DNAse and released the binding proteins

51
Q

What are PERMISSIVE transcription factors?

A

General Transcription factors!!

Transcription factors which DO NOT regulate gene expression but are necessary for all transcription as they bind at the promoter or interact with the TIC to initiate transcription

52
Q

What are SPECIFIC or REGULATORY transcription factors?

A

Activators!! Repressors!!

Bind anywhere around the gene (upstream/downstream of promoter) to regulate (increase or decrease) the transcription of a gene

Bind to enhancers or silencers
Or to a REGULATORY COMPLEX

53
Q

What is special about some specific/regulatory transcription factors?

A

They can act as BOTH an activator and a repressor

54
Q

How do regulatory transcription factors regulate DNA transcription? (3 ways)

A

1) Interact with the RNA pol II complex
2) Alter acetylation of DNA
3) Bind to other transcription factors to form complexes

55
Q

Where do regulatory TFs bind and how is this different to permissive TF?

A

Regulatory bind anywhere around the gene (upstream or downstream of the promoter)
- Bind to enhancers, silencers, or to a regulatory complex

Permissive only bind at promotors or to TIC

56
Q

Why must DNA loop?

A

To allow the interaction of distant binding proteins (regulatory proteins) which must physically interact with the transcription complex

57
Q

What distance away should proteins be in order for them to be able to interact and why?

A

Directly neighbouring
OR
>500bp apart

As it is hard for chromatin to bend
After this, chances of finding the binding site is lower

58
Q

Which genes do TF bound to enhancers activate?

A

Any gene within a region that they are bound to - not specific (they are promiscuous)

59
Q

What are enhancers?

A

Binding sites on DNA for transcriptional ACTIVATORS

60
Q

What are silencers?

A

Biding sites on DNA for transcriptional REPRESSORS

61
Q

What 2 things can block the promiscuous action of transcriptional activators?

A

1) Insulator elements
2) Barrier sequences

Block the activator from activating genes inappropriately

62
Q

How is the transcription of a single gene modulated?

A

1) Complexes (strongly activating/inhibiting, weakly activating/inhibiting) receive inputs from within the cell and from outside the cell
2) All inputs read by a DNA code - all have influence at the promoter about HOW MUCH the gene should be expressed
3) Combination of these inputs determine if the gene is expressed
- INPUTS are referred to as ‘genetic switches’
- Each genetic switch responds to intrinsic and extrinsic regulation
- Genetic switches work together to achieve an output

63
Q

Describe tryptophan as a genetic switch

A

Low levels of tryptophan:
- Tryptophan genes turned on

High levels:
- Tryptophan binds to TF with regulates tryptophan synthesis - represses the transcription synthesis genes

64
Q

What are 7 ways to regulate a transcription factor in eukaryotes?

A

1) Protein synthesis
2) Ligand binding
3) Protein phosphorylation
4) Addition of a second subunit (activation subunit)
5) Unmasking (remove inhibitory protein)
6) Stimulation of nuclear entry (remove inhibitory protein)
7) Release from the membrane (cleavage)

65
Q

How might one TF help another?

A
  • Make a dimer
  • Prevent ‘falling off’ the DNA
  • Binding of one TF to DNA may allow the binding of another TF
66
Q

How might the binding of one TF to DNA allow a different TF to bind?

A

The first TF may unwind the DNA slightly to reveal another binding site

67
Q

What is a ‘logical network’?

What does it show?

A

A mathematical model of the interactions between transcription factors

Shows that depending on what TF work together - TFs work together to give an output

68
Q

What 3 ways might TF work together to activate/inbhibit others?

A

1) Positive feedback (upregulate itself)
2) Negative feedback (downregulate itself)
3) Flip-flop (2 TF inhibit eachother)
4) Feed-forward loop (activate a chain of TFs)

BMS 242 - Advanced Cell and Molecular (24 decks)

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