CMB2001 Exam Questions With Verified
Answers.
Core Promoter Elements - answerTATA box = repetitive sequence of thymine and adenine
Initiator (INR) = sequence of pyramidines like thymine and cytosine
DPE = downstream promoter elements found along the DNA
Common to have >1 promoter but not al...
Core Promoter Elements - answer✔✔TATA box = repetitive sequence of thymine and adenine
Initiator (INR) = sequence of pyramidines 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 are able to 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 distalling 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; 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 the 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 antirepressing function
TFIIB - answer✔✔Recruits RNA polymerase II and TFIIF so is important for start site selection
TFIID - answer✔✔Binds TATA box in the core promoter region and recruits TFIIB
It has a trisobular structure = rounded triangle
Made of TATA binding proteins (TBPs) and TBP associated factors (TAFs). TBPs can act alone
but not on 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
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 aspartamine 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
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