Promoter Structure and Function Flashcards
regulator elements/molecules act – affecting expression of a gene in the same chromosome
in cis
What are the 4 cis-regulatory modules?
- promoter
- enhancer
- silencer
- insulator
what is a promoter?
what does it consist of? where is it found?
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
What is an enhancer?
DNA seq that causes an increase in gene expression
- acts independently of position and orientation with respect to the gene
What is a silencer?
DNA sequence that causes a decrease in gene expression
What is an insulatory?
DNA seq that blocks promoter activation by an enhancer
enh |(insulator) <promoter
What is the RNAPII Core Promoter?
how long is it?
minimal set of sequence elements for accurate transcription initiation by Pol II machinery in vitro
usually 60 nucleotides |
what are the 4 important elements of RNAPII Core Promoter?
- RNA Polymerase binding site
- TATA box
- transcription start site
most proximal to start codon AUG
What is the TFIIB recognition element?
G-C rich
- extension of a subset of TATA boxes
What is DPE?
downstream promoter element
- redundancy for genes containing TATA box
- found in ‘TATA-less’ promoters
What is the eukaryotic transcription initiation complex?
- RNAPII
- 5 GTFs
What are the steps of pre-initiation complex (PIC) assembly?
- TATA box binding protein (TBP) component of TFIID binds to TATA box of the promoter
- TFIIA and TFIIB bind to TFIID to stabilize
- TFIIF binds to RNAPII which then binds to TATA box complex
- TFIIE recruits TFIIH which are both added to the complex
- helicase + ATP induces open promoter complex
What are the steps after PIC assembly?
- TFIIH subunit melts DNA at the promoter (with energy from ATP hydrolysis)
- RNAPII starts transcribing RNA while another TFIIH subunit phosphorylates Ser 5 and 7 of CTD of RNAPII
- RNAPII is now highly processive
- all GTFs dissociate except TBP after Pol II moves
What are the two expression states of RNAPII promoter?
- basal transcription: requires RNAPII + GTFs IID, IIB, IIE, IIF, and IIH (IIA is variable in vitro and essential in vivo)
- activated transcription: transcriptional direct or indirect activators
what are transcriptional activators? What do they do? Where do they bind?
increase local [ ] of GTPs to increase efficiency and frequency of initiation
- bind to upstream proximal promoter elements (UAS) or distal enhancers
Promoter proximal TFs function in a –, –, –, manner with respect to the core promiter
distance, location and orientation-specific
enhancers can act independent of –, –, –, with respect to target gene
orientation, distance, and location
2 purposes of enhancers?
- 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)
What is a possible explanation for why enhancers operate independent of position and orientation?
- 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
Why is there so many additional proteins, like 1 million Da mediator, required for transcription initiation?
DNA template packaged into chromatin in vivo
How and when does RNAPII pausing occur?
- 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
How does the enhancer help with PIC and transcription initiation and transcription elongation?
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
Transcription occurs in – –
short bursts of initiation events + inactivity
what increases burst size and frequency?
high number of Pol II bound to core promoter increases burst size
enhancers increase burst frequency
What is a multiple cloning site (MSC)?
region of a plasmid that contains restriction enzyme cut sites
What are examples of reporter genes
- Green fluoresnce based reporter
- lacZ gene makes B-Galactosidase coloured product
- luciferas
What would be included in a vector?
- Amp selectable marker
- ori
- reporter gene
- MSC
- synthetic polyA signal for ori and for reporter gene
what is a vector used for?
transfection assay: measure gene expression in vivo
enhancer activity is measured in — to test the ability to activate transcription at distal core promoter and drive reporter gene expression
reporter assays
check upstream and downstream of promoter
core promoter activity can be measure with — to measure ablity to initiate transcription in response to enhancer
reporter assay
check with enhancer and w/out enhancer
promoter activity is defined as the ability to
drive transcription autonomously (core promoter + proximal promoter/UAS) activity
What are the 4 methods to detect sequence specific binding of proteins to DNA?
- DNaseI footprinting
- methylation interference/protection assays
- electrophoretic mobility shift assays
- chromatin immunoprecipitation
What is the purpose of DNase I footprinting? How does it work? What does it tell you?
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
What is the purpose of a methylation interference assay?
- identifies As and Gs in the binding site that, when methylated, interference with protein binding
How does a methylation interference assay work?
- labelled DNA
- use DMS to methylate at Gs (or As)
- add nuclear extract which has proteins
- specific proteins bind in their binding sites on DNA
- if methylated in binding site = protein can’t bind = appears on gel
- if methylated not in binding site = protein can bind = protein interferes with visualization = gap in gel
How is methylation interference assay better than DNase I footprinting?
-CH3 is smaller
better resolution because can get closer to DNA binding region
What does an electrophoretic mobility shift assay do? EMSA
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
What is the process of Chromatin Immunoprecipitation Assay CHIP?
- cross link a protein to DNA
- isolate chromatin and shear DNA (cut into small pieces)
- precipitate chromatin with prptein specific antibody
- reverse cross-link and digest protein
- ligase P1 and P2 adaptors to sequence to construct fragment library
- sequence and characterize
understand proteins bound to DNA
How does a transfection assay work?
- make two plasmids: one with a target gene X and one with reporter gene + target gene X binding site
- transfect into cell
- 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
