Transcriptional Control of Gene Expression 1 (L10) Flashcards
CAP
catabolite activator proteins - bind recognition site and stabilize binding of RNA pol to promoter -> increases transcription
what does CAP depend on?
needs cAMP to bind
sigma factor
an IF - different sigma factors used for transcribing different genes
two functions of enhancers
- spatial-temporal regulation of gene expression
2. alteration of expression level
how do enhancers work?
by stabilizing RNA pol II binding via mediator proteins
is an enhancer’s location important for its function?
not often
how do enhancers bind regulatory proteins?
multiple recognition sequences - i.e. TFs (or activators/repressors)
what does the proximal/basal promoter always bind?
always bind same TFs
what does promoter consist of?
enhancers + basal promoters (DNA)
what do activators consist of?
protein
difference b/w enhancers and proximal promoter
enhancers can be all over; prox promoter is right before the gene
UAS
upstream activating sequence - in yeast, equivalent of enhancer
mammalian numbers of promoters and enhancers
only one promoter but multiple enhancers
spatial-temporal regulation
control where genes are expressed and when expressed during life cycle
what are enhancers?
regulatory sequences (or promoter elements) that are bound by TFs - aid in signal transduction
TFBS
TF binding site - in one enhancer, can have many TFs binding at same TFBS
TATA box
consensus sequence - same sequence found more often than just randomly would occur - important regulatory sequence for RNA pol
CpG island
20-50 nts within 100 bps upstream
functions of RNA pol I
- ribosome components
- protein synthesis
- binds basal promoter elements
functions of RNA pol II
- transcribes mRNA, miRNA, siRNA
- encodes protein
- RNA splicing
- post-translational gene control
functions of RNA pol III
- protein synthesis
- ribosome component, protein synthesis
- RNA splicing
- signal-recognition particle for insertion of polypeptides into the ER
- various unknown functions
CTD
C-terminal domain on beta1 subunit of RNA pol II - consists of 52 repeats of Tyr-Ser-Pro-Thr-Ser-Pro-Ser (phosphorylation)
how can you differentiate when CTD is phosphorylated or not?
phosphorylated form = red -> more decondensed areas
dephosphorylated form = green
both = yellow
polytene chromosome
giant chromosome used to study regulation of gene expression
experimental definition of promoter elements - two ways
- deletion analysis
2. linker scanning mutation/deletion
deletion analysis
Chop promoter into smaller pieces or use probes to replicate smaller pieces.
1. use recombinant DNA techniques to create a 5’ deletion series.
2. Ligate into vector carrying reporter gene.
3. Transform into E. coli and isolate plasmid DNAs.
4. Transfect each type of plasmid (1-5) separately into cultured cells.
5. Prepare cell extract and assay activity or reporter enzyme.
Reporter gene expression levels tell you which areas are necessary for expression.
what does deletion analysis tell you?
where important regions of promoter are - useful for analyzing basal promoter region
difference b/w deletion analysis and linker scanning mutation/deletion analysis
deletion: chops from end
linker: chops in middle in overlapping way
linker scanning mutation/deletion
same principles as deletion analysis, but chops in middle in overlapping way - allows for better determination of where the important elements are
what happens if you delete the TATA box?
RNA pol can’t bind at all - no expression
what happens if you delete other regulatory elements than the TATA box?
these just help stabilize binding of RNA pol, so if deleted, just get a decrease in expression
EMSA
electrophoretic mobility shift assay/gel shift
1. mix DNA probe with protein fractions from chromatography
2. run on gel
If protein does NOT bind probe, runs faster.
If protein DOES bind probe, lower mobility - slower migration. Any lanes with slow migrating bands - know there is a protein in this fraction that can bind promoter
what does yeast-1-hybrid test?
interaction b/w protein and DNA - if protein from cDNA library can bind to promoter element -> get expression of that construct
how do you verify potential identification of DNA-binding proteins?
- Purify the protein by sequence-specific DNA affinity chromatography.
- Sequence protein and clone the gene.
- Insert a plasmid containing the gene sequence for what you think is protein X and a plasmid containing a reporter gene and a binding site for protein X into a cell.
- If the gene produces protein that binds to the X-binding site, then transcription of the reporter gene will occur (means your protein truly binds the promoter)
difference b/w yeast-1-hybrid and yeast-2-hybrid
1: 1 construct, tests protein-DNA interaction
2: 2 constructs (bait/prey), tests protein-protein interaction
describe expression when you delete parts of AD
less expression than normal
describe expression when you delete all of AD
no expression
how can you define DBD and AD?
deletion mutants - N and C terminal deletion mutants or internal deletion mutants
functional domains are…
modular
how can TF’s be regulated?
some have activation domains, some have repression domains
