Promoter Structure and Function

Question 1

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

Answer 1

in cis

Question 2

What are the 4 cis-regulatory modules?

Answer 2

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

Question 3

what is a promoter?

what does it consist of? where is it found?

Answer 3

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

Question 4

What is an enhancer?

Answer 4

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

Question 5

What is a silencer?

Answer 5

DNA sequence that causes a decrease in gene expression

Question 6

What is an insulatory?

Answer 6

DNA seq that blocks promoter activation by an enhancer

enh |(insulator) <promoter

Question 7

What is the RNAPII Core Promoter?

how long is it?

Answer 7

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

usually 60 nucleotides |

Question 8

what are the 4 important elements of RNAPII Core Promoter?

Answer 8

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

most proximal to start codon AUG

Question 9

What is the TFIIB recognition element?

Answer 9

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

Question 10

What is DPE?

Answer 10

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

Question 11

What is the eukaryotic transcription initiation complex?

Answer 11

• RNAPII
• 5 GTFs

Question 12

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

Answer 12

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

Question 13

What are the steps after PIC assembly?

Answer 13

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

Question 14

What are the two expression states of RNAPII promoter?

Answer 14

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

Question 15

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

Answer 15

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

Question 16

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

Answer 16

distance, location and orientation-specific

Question 17

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

Answer 17

orientation, distance, and location

Question 18

2 purposes of enhancers?

Answer 18

• 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)

Question 19

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

Answer 19

• 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

Question 20

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

Answer 20

DNA template packaged into chromatin in vivo

Question 21

How and when does RNAPII pausing occur?

Answer 21

• 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

Question 22

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

Answer 22

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

Question 23

Transcription occurs in – –

Answer 23

short bursts of initiation events + inactivity

Question 24

what increases burst size and frequency?

Answer 24

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

enhancers increase burst frequency

Question 25

What is a multiple cloning site (MSC)?

Answer 25

region of a plasmid that contains restriction enzyme cut sites

Question 26

What are examples of reporter genes

Answer 26

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

Question 27

What would be included in a vector?

Answer 27

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

Question 28

what is a vector used for?

Answer 28

transfection assay: measure gene expression in vivo

Question 29

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

Answer 29

reporter assays | check upstream and downstream of promoter

Question 30

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

Answer 30

reporter assay | check with enhancer and w/out enhancer

Question 31

promoter activity is defined as the ability to

Answer 31

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

Question 32

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

Answer 32

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

Question 33

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

Answer 33

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

Question 34

What is the purpose of a methylation interference assay?

Answer 34

- identifies As and Gs in the binding site that, when methylated, interference with protein binding

Question 35

How does a methylation interference assay work?

Answer 35

1. 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

Question 36

How is methylation interference assay better than DNase I footprinting?

Answer 36

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

Question 37

What does an electrophoretic mobility shift assay do? EMSA

Answer 37

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

Question 38

What is the process of Chromatin Immunoprecipitation Assay CHIP?

Answer 38

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

Question 39

How does a transfection assay work?

Answer 39

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 ## Footnote 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