27 regulation Flashcards
why is regulation of gene expression required?
- only a fraction of genes are needed at a given time for the cell to perform specific tasks (requirement for gene products changes over time and environmental conditions)
- Optimal use of available energy and resources, since the cost of protein synthesis is high
What are the levels of transcriptional and translational regulation?
Transcriptional
* synthesis of mRNA
* splicing
* mRNA stability
Translational
* microRNA
What controls transcription in bacterial cells?
operons
What do operon consist of?
promoter + operator + structural gene
Where can regulator protein bind on a operon?
on the operator to decide on or off for transcription
what is the purpose of promoter in an operon?
for binding of RNA polymerase to initiate transcription
what is the purpose of operator in an operon?
to regulate transcription, binding of regulator protein to operator controls transcription
what is the purpose of structural genes in an operon?
encoding proteins, to be transcribed
what are the 2 operon systems in prokaryotes?
- inducible system - operon normally not transcribed, inducer turns on transcription (e.g. lac operon)
- Repressible systems: operon normally transcribed, products of a pathway turn off transcription (e.g. try operon)
what are examples of inducible system and repressible systems?
inducible - lac operon
repressible - trp operon
what is E.coli food source?
glucose as primary food source
lactose as an laternative food source
What 2 enzymes (proteins) are required if lactose is present in E.coli from what gene?
2 enzymes (i.e. galactoside permease from LacY gene and beta-galatosidase from lacZ gene) is required for uptake and metabolism of the lactose are expressed
if no lactose, levels of these proteins are very low
what does galactoside permease and beta-galactosidase do?
galactoside permease - facilitate import of lactose from outside through this permease and into the cell
beta-galactosidase - break down into gluclose and galactose
what is lac operon?
a negative inducible operon
what happens in the lac operon in the absence of lactose?
- active repressor binds to operator sequence in operon and repress it
- RNA polymerase wont be able to bind to promotor -= no activativation LacY and Z = no enzyme produced cuz no transcription of operon
What happens in lac operon when there is presence of lactose?
- in cell theres a basal (low) level of beta galactosidase - detect lactose and converts it into allolactose (inducer)
- allolactase is a signal that binds to repressor protein to make it go away from the operon
- so now RNA polymerase can bind to the promoter and allow transcription and translation of LacZ and Y to produce β-galactosidase and permease
How does glucose level control the lac operon
- glucose levels regulate cAMP levels
- high glucose inhibit conversion of ATP to cAMP
high glucose = low cAMP
low glucose = high cAMP
What is cAMP needed for?
Transcription
combine with CAP protein and bind to promotor to activate transcription of lac operon
what is the acitvity of lac operon when there is glucose but no lactose?
glucose present = low cAMP
no lactose = repressor binds
= no lac mRNA
whats the activity of lac operon when there is no glucose and lactose is present?
no glucose = high cAMP
cAMP bind with CAP protein to initiate lac mRNA
inducer also binds to repressor
= abundant lacmRNA
what is the activity of lac operon when glucose and lactose are present?
glucose = low cAMP
Low cAMP = no binding with CAP protein = no active CAP-cAMP complex to initiate transcription
even though inducer removes repressor
how does lac operon show positive and negative control?
repressor - negative control (supress)
CAP and cAMP - positive control (enhance)
What is CAP that binds to cAMP?
a regulatory protein (catabolite activator protein)
Where does CAP bind to?
binds to cAMP. the complex binds to DNA just upstream of promoter resulting in a more efficient binding of RNA polymerase to the promoter and activate transcription (positive regulation)
What is catabolite repression?
presence of glucose lowers the cell conc of cAMP, thus less CAP binding to promoter = less efficient transcription of lac operon
What is an example of negative repressible operon?
trp operon
What does trp operon control the synthesis of?
tryptophan
operon is shut off when tryptophan is abundant
= a repressible system
repressible vs inducible system
when there is something = induce the operon
when there is somthing = repress the operon
compare regulatory protein of trp operon and lac operon
lac - the regulatory protein is synthesized as an active repressor (on unless someting binds to it)
trp - the regulatory protein is synthesized as an inactive repressor (off unless something binds to it)
How does an inactive repressor become active?
when it binds with a corepressor (trypotophan in trp operon)
Whats the activity of trp operon in the absence of a corepressor?
when tryptophan is absent, the inactive repressor cannot bind to operator = transcription of operon
Whats the activity of trp operon in the presence of a corepressor?
repressor binds to operator = block transcription of operon
How can gene expression be controlled in eukaryotes?
transcription factor-DNA binding
How do transcription factors (TF) bind DNA?
TF will recognize DNA sequence and will bind to major groove of DNA (more open)
minor groove is too narrow for protein
How do TFs contact DNA grooves?
through H bond, ionic bond, and hydrophobic interactions
TF have a side chain of AA and grabs the DNA and tries to interact
What is the common shape found in DNA binding proteins/domains?
Zinc Finger protein family
- has a alpha helix and beta sheet
can fit very well in terms of shape and allow side chain to interact with AGCT
why are there certain specific configuration in order to bind to DNA?
need to fit protein into the groove
Besides Zinc finger protein family, what is another type of shape of DNA binding protein/domain?
Helix-turn-helix (HTH) motif
2 helixes
recognition helix fits into major groove
other helix helps maintain angle
What is the Multimeric combinations of TF?
assembly of multiple protein subunits to form a functional unit that can regulate gene expression.
can activate or inhibit the transcription of that DNA
How do we know how strongly the TF binds to each binding site?
using logo plots
* sequence logos capture binding site variants, and represent both base frequency and conservation of each position
How do we measuring affinity?
using protein binding arrays
* each spot has different oligo sequence
* oligos capture all variants of binding sequence
* add TF and let bind to each oligo
* quantify TF bound using fluorescent antibody
* repeat for many TFs
synthesize all the possible short sequences and you label it
then show your TF of interest and see which one it binds to
if it doesnt bind it just washes away
then you sequence the ones that bind
What do TFs in the same family have in common and different?
have common strongest affinity sequence
but diffrent weaker affinity sequences
why do domains have differnt affinities for diff motifs?
allow TF to interact with mutltiple motifs
Why do we want to know the affinity of TF?
Scientists are characterizing which TF will bind to which pieces of DNA to turn on or off the transcription to facilitate gene regulation
Interpret this
2 diff TF
Both can bind to GGGTCA
But only HNF4a can bind to GGTCCA (blue)
Greater the signal the better the binding
At what stage can gene expression be controlled?
at multiple levels
How can microRNA regulate gene expression in eukaryotic cells?
microRNA - double stranded precursor RNA, + Si RNA bind to dicer (endonuclease protein that cuts DNA into short segments)
