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CMB 2001 EXAM STUDY SET LATEST UPDATED
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CMB 2001 EXAM STUDY SET LATESTUPDATED
Core Promoter Elements - ANSWER TATA box = repetitive sequence of thymine
and adenine
Initiator (INR) = sequence of pyrimidines like thymine and cytosine
DPE = downstream promoter elements found along the DNA
Common to have >1 promoter but not all of them
CpG islands - ANSWER Sequences high in CG frequency. They can escape
methylation which would lead to gene silencing. Demethylation would lead to
over-expression of genes.
Regulatory Promoter Elements - ANSWER UAS and URS found proximal
upstream; silence and repressor found distilling upstream
UAS + activator = Activator binding site
URS + silencer = Repressor binding site
Identifying promoter elements - ANSWER 1. Sequence comparison -
high-frequency base at particular point = consensus sequence
2. Reporter Gene Assay using GFP, luciferase, lac-Z; the amount of reporter is the
amount of gene expressed
5' deletion series - ANSWER Otherwise known as protein bashing:
1. Recombinant DNA techniques applied
2. Ligate vector carrying reporter gene
3. Transform E.coli and isolate plasmid DNA
4. Transfect each type of plasmid and culture cells
,5. Prepare cell extract and carry out reporter assay which will show the effect of the
deletion
Reporters are used to identify - ANSWER - When a gene is expressed
- Where a gene is expressed
- What signal causes it's expression
- What factors and sequences are involved
RNA polymerase I - ANSWER Found in the nucleolus produces rRNA such as
28S, 18S and 5.8S
RNA polymerase II - ANSWER Found in the nucleus and produces mRNAs
including siRNA and miRNAs
RNA polymerase III - ANSWER Found in the nucleus and produces tRNA
How is prokaryotic RNA polymerase II different to eukaryotic RNA polymerase
II? - ANSWER Prokaryotic = 2 x alpha, beta, beta', omega subunits
Requires sigma cofactor to recognise DNA
Eukaryotic = much more complex. Has 12 subunits (some which are similar to the
prokaryotic subunits) and sigma cofactor is replaced by Transcription factors which
will recruit RNA polymerase II to the promoter site
The sequence of TFII binding - ANSWER D -> A -> B -> F + RNA polymerase II
-> E + H
CTD on RNA polymerase II - ANSWER C-terminus domain on beta' subunit is
highly phosphorylated during transcription
TFII fate during transcription - ANSWER - TFII D and A remain at the promoter
site
- TFIIF remains with the RNA polymerase II
- TFII H, E and B
TFIIA - ANSWER Promotes binding of TFIID and has anti repressing function
,TFIIB - ANSWER Recruits RNA polymerase II and TFIIF so is important to start
site selection
TFIID - ANSWER Binds TATA box in the core promoter region and recruits
TFIIB
It has a triangular structure = a rounded triangle
Made of TATA binding proteins (TBPs) and TBP-associated factors (TAFs). TBPs
can act alone but not in TATAless region
TFIIE - ANSWER Recruits TFIIH and modulates it's activity
TFIIH - ANSWER Helicase activity due to containing a helicase. XPB and XPD
regions cause promoter melting. Contains CORE and CAK - separate and assists in
cell cycle regulation
TFIIH involved in DNA repair
UAS affects transcription by - ANSWER increasing rate
Common Enhancer Elements - ANSWER They are constituitively active and often
found proximal to promoter region:
1) GC box = GGGCCC i.e. Sp1
2) Octamer = ATTTGCAT i.e. Oct-1
3) CAAT = GGCCAATCT i.e. NF-1
Response Elements - ANSWER Their activity is regulated by stimuli:
1) HSE = binds heat shock factor in response to heat shock
2) SRE = binds serum response factor in response to serum
Modular - ANSWER Functional alone but may be combined to form groups
DNA binding domains - ANSWER Zinc finger domain, leucine zipper domain,
helix loop helix, homeodomain
Activator domain for eukaryotic transcription - ANSWER Determined by their
amino acid composition. Lack sequence conservation and generally unstructured,
, contain multiple short segments and work in additive fashion
Acidic patch (-ve residues mainly aspartame and glutamine), glutamine rich (Sp1)
and proline rich
Methods of finding activation domains - ANSWER In vivo -> 1) Reporter Assays
In vitro -> 1) DNA footprinting
2) Electrophoretic Mobility Shift Assay (EMSA)
3) Transcription Assay
Electrophoretic mobility shift assay - ANSWER DNA protein complex is run on
non denaturing acrylamine gel and free DNA is separated from complex. It
measures the ability of a protein (activator) to bind to sequence (DNA) which is
often radiolabelled
Transcription Assay - ANSWER DNA binding domain and activating domain is
required for transcription. Truncated mutant forms are tested to see where the DNA
binding domain is and where activator binding domain is.
Measuring Gene Expression with Microarrays - ANSWER Control and RNA
lacking transcription factors is run on labelled microarrays which show how
transcription factors affect transcription. Transcription factors may be activating or
repressing.
Measuring Gene Expression with ChIP - ANSWER ChIP = Chromatin
Immunoprecipitation
1) Protein crosslinked trapping DNA-protein complex
2) Shear DNA through technique such as sonication
3) Purify activator sites by binding a protein specific antibody
4) Reverse crosslinks to remove protein
5) Interrogate DNA by PCR, Microarrays (ChIP on ChIP) or sequencing
(ChIP-seq) to identify activator binding sequences
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