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3382 > Promoter Structure and Function > Flashcards

Promoter Structure and Function Flashcards

1
Q

regulator elements/molecules act – affecting expression of a gene in the same chromosome

A

in cis

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2
Q

What are the 4 cis-regulatory modules?

A
  1. promoter
  2. enhancer
  3. silencer
  4. insulator
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3
Q

what is a promoter?

what does it consist of? where is it found?

A

DNA seq required for initiation of transcription
- consists of ‘core promoter’ and proximal regulatory elements
- almost always at 5’ end of gene, upstream of transcription start site

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4
Q

What is an enhancer?

A

DNA seq that causes an increase in gene expression
- acts independently of position and orientation with respect to the gene

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5
Q

What is a silencer?

A

DNA sequence that causes a decrease in gene expression

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6
Q

What is an insulatory?

A

DNA seq that blocks promoter activation by an enhancer

enh |(insulator) <promoter

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7
Q

What is the RNAPII Core Promoter?

how long is it?

A

minimal set of sequence elements for accurate transcription initiation by Pol II machinery in vitro

usually 60 nucleotides |

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8
Q

what are the 4 important elements of RNAPII Core Promoter?

A
  • RNA Polymerase binding site
  • TATA box
  • transcription start site

most proximal to start codon AUG

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9
Q

What is the TFIIB recognition element?

A

G-C rich
- extension of a subset of TATA boxes

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10
Q

What is DPE?

A

downstream promoter element
- redundancy for genes containing TATA box
- found in ‘TATA-less’ promoters

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11
Q

What is the eukaryotic transcription initiation complex?

A
  • RNAPII
  • 5 GTFs
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12
Q

What are the steps of pre-initiation complex (PIC) assembly?

A
  1. TATA box binding protein (TBP) component of TFIID binds to TATA box of the promoter
  2. TFIIA and TFIIB bind to TFIID to stabilize
  3. TFIIF binds to RNAPII which then binds to TATA box complex
  4. TFIIE recruits TFIIH which are both added to the complex
  5. helicase + ATP induces open promoter complex
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13
Q

What are the steps after PIC assembly?

A
  1. TFIIH subunit melts DNA at the promoter (with energy from ATP hydrolysis)
  2. RNAPII starts transcribing RNA while another TFIIH subunit phosphorylates Ser 5 and 7 of CTD of RNAPII
  3. RNAPII is now highly processive
  4. all GTFs dissociate except TBP after Pol II moves
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14
Q

What are the two expression states of RNAPII promoter?

A
  1. basal transcription: requires RNAPII + GTFs IID, IIB, IIE, IIF, and IIH (IIA is variable in vitro and essential in vivo)
  2. activated transcription: transcriptional direct or indirect activators
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15
Q

what are transcriptional activators? What do they do? Where do they bind?

A

increase local [ ] of GTPs to increase efficiency and frequency of initiation
- bind to upstream proximal promoter elements (UAS) or distal enhancers

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16
Q

Promoter proximal TFs function in a –, –, –, manner with respect to the core promiter

A

distance, location and orientation-specific

17
Q

enhancers can act independent of –, –, –, with respect to target gene

A

orientation, distance, and location

18
Q

2 purposes of enhancers?

A
  • increase rate of transcription initiation in all cells in a population
  • increase probability that a gene will be in a transcriptionally competent site (open core promoter histone for access to factors)
19
Q

What is a possible explanation for why enhancers operate independent of position and orientation?

A
  • DNA can loop so that activator-enhancers OR repressor-silenceers are close to target (PIC or GTFs)
  • operate through intermediary called co-activator/mediator
  • then, size of loop (distance) or orientation (5’-3’) doesn’t matter
20
Q

Why is there so many additional proteins, like 1 million Da mediator, required for transcription initiation?

A

DNA template packaged into chromatin in vivo

21
Q

How and when does RNAPII pausing occur?

A
  • after RNAPII escapes TSS, it synthesizes a short stretch of nascent RNA then pauses downstream of TSS
  • DSIF and NELF binds to RNAPII and nascent RNA for pausing
  • CDK9 phosphorylates CTP of RNAPII so it starts productive elongation
22
Q

How does the enhancer help with PIC and transcription initiation and transcription elongation?

A

PIC: TFs and cofactors (COFs) bind to enhancer – TFs and COFs directly interact with GTFs and RNAPII

initiation: recruits mediation complex (MED) for PolII dissociating from GTFs and TSS

elongation: recruits cofactors that affect pause release or recruit and stimulate CDK9

23
Q

Transcription occurs in – –

A

short bursts of initiation events + inactivity

24
Q

what increases burst size and frequency?

A

high number of Pol II bound to core promoter increases burst size

enhancers increase burst frequency

25
Q

What is a multiple cloning site (MSC)?

A

region of a plasmid that contains restriction enzyme cut sites

26
Q

What are examples of reporter genes

A
  1. Green fluoresnce based reporter
  2. lacZ gene makes B-Galactosidase coloured product
  3. luciferas
27
Q

What would be included in a vector?

A
  1. Amp selectable marker
  2. ori
  3. reporter gene
  4. MSC
  5. synthetic polyA signal for ori and for reporter gene
28
Q

what is a vector used for?

A

transfection assay: measure gene expression in vivo

29
Q

enhancer activity is measured in — to test the ability to activate transcription at distal core promoter and drive reporter gene expression

A

reporter assays

check upstream and downstream of promoter

30
Q

core promoter activity can be measure with — to measure ablity to initiate transcription in response to enhancer

A

reporter assay

check with enhancer and w/out enhancer

31
Q

promoter activity is defined as the ability to

A

drive transcription autonomously (core promoter + proximal promoter/UAS) activity

32
Q

What are the 4 methods to detect sequence specific binding of proteins to DNA?

A
  1. DNaseI footprinting
  2. methylation interference/protection assays
  3. electrophoretic mobility shift assays
  4. chromatin immunoprecipitation
33
Q

What is the purpose of DNase I footprinting? How does it work? What does it tell you?

A

type of assay that identifies regions that are protected from nuclease digestion which are priten binding regions
- DNase I = nuclease
- if cut, appears on gel
- if protected by some binding protein, gap in gel
- tells you where protein binding location is (size)

Example: a lot of TBP or TFIID = larger gap in gel

34
Q

What is the purpose of a methylation interference assay?

A
  • identifies As and Gs in the binding site that, when methylated, interference with protein binding
35
Q

How does a methylation interference assay work?

A
    1. labelled DNA
  2. use DMS to methylate at Gs (or As)
  3. add nuclear extract which has proteins
  4. specific proteins bind in their binding sites on DNA
  5. if methylated in binding site = protein can’t bind = appears on gel
  6. if methylated not in binding site = protein can bind = protein interferes with visualization = gap in gel
36
Q

How is methylation interference assay better than DNase I footprinting?

A

-CH3 is smaller
better resolution because can get closer to DNA binding region

37
Q

What does an electrophoretic mobility shift assay do? EMSA

A

add radiolabelled nucleic acid with nuclear extract
- see what shifts down the gel fastest to see if proteins bind
- supershift complex is due to antibody binding

38
Q

What is the process of Chromatin Immunoprecipitation Assay CHIP?

A
  1. cross link a protein to DNA
  2. isolate chromatin and shear DNA (cut into small pieces)
  3. precipitate chromatin with prptein specific antibody
  4. reverse cross-link and digest protein
  5. ligase P1 and P2 adaptors to sequence to construct fragment library
  6. sequence and characterize

understand proteins bound to DNA

39
Q

How does a transfection assay work?

A
  1. make two plasmids: one with a target gene X and one with reporter gene + target gene X binding site
  2. transfect into cell
  3. protein X will bind to binding site and you will see reporter gene

see if a protein will bind to a specific binding site | see if TF binds to a TFBS or to any region of the DNA

3382 (20 decks)

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