Chapter 6: Regulation of Gene Expression Flashcards
-/+ Regulation
2 sites?
Negative regulation is system is by default off and there needs to be a signal (inducer) to turn it on, presence of lactose in medium is an inducer.
Positive regulation, by default system is on and the signal leads to the turn off of the system, tryptophan protein is example.
Activators/ repressors exert their actions through two alternative sites: Allost eric site or DNA binding site.
The lactose operon
A repressor protein (produced by the lac i gene) binds to an operator sequence (O) blocking transcription of genes lac z, lac y and lac a. Lactose induces transcription. POZYA is an operon(a genetic unit of coordinated expression)
lac mutants
lac z = structural gene for β-galactosidase. The enzyme hydrolyses β-galactosides in general.
lac y =mutants show structural gene for β-galactoside permease. The protein allows for the transportation of lactose and β-galactosides across the cell membrane.
lac i = I is repressor, codes for repressor protein that likes to bind to the operator and turn it off in the absence of lactose (if + the operon is off since the repressor protein will be sitting on it so nothing will happen) . A constitutive mutant (always expresses genes).
Other mutants also show constitutive expression (lac o-, always on) even in the presence of an active repressor.
Cis-regulation: Operator mutants. Trans regulation: lac i mutants
(O+ Z+ Y+/ F’ Oc Z+ Y+) result shows O cis dominant to O+Last two show O is cis acting.
The last two show that I+ is dominant to I-and is trans acting.
Catabolic repression
- A catabolic product of glucose prevents induction by lactose catabolite repression
- When glucose has a high concentration, the cAMP concentration will be low.
- Catabolite repression is mediated by a catabolite activator protein (CAP) and cAMP. High glu inhibits adenylate cyclase –>low cAMP.
- The lac promoter has a CAP-cAMP binding site.
- Binding of CAP-cAMP to the promoter is needed for lac operon induction (positive control
- CAP (catabolite activator protein) made by the gene known as crp.
- Of course, mutants that are anything other than the normal lac operon, cannot convert ATP into cAMP and thus cannot produce β-galactosidase because of cAMP concentration not big enough.
Footprinting/Gel Mobility Assay (lac operon)
Foot printing (Labeled DNA + protein binding reaction. Add DNAse and analyze fragments in a gel) Gel mobility assay: (Labeled DNA + protein binding reaction. Labeled DNA without protein. Run in a gel)
tryptophan operon
high tryptophan binds (allosteric site) activates a repressor. low tryptophan —>repressor inactive —>transcriptopn of enzymes needed to make tryptophan. Attenuation: If high tryp —> rapid translation—>termination of transcription—>no more Trp. if low Trp, translation stalls—>transcription continues
Lambda phage
Lysogeny: CI —>prepressor protein—>blocks promoters for transcription og lytic pathway genes .
Lytic: CI—>RNApol—>Cro—>blocks promoter for CI transcription + antiterminaotr proteins allow progression of transcription.
Under poor conditions—> Ciii—>Cii Cii—>recuit polymerase for: Cro amtisease; CI sense—>lysongeny.
Good conditions —>CIII—>CII is degrated—>RNApol transits Cro—>lysis
Cis-acting target sequences
- Core promoter: TATA box (TATA helps position RNA polymerase for transcription initiation)
- Promoter proximal elements (needed by core promoter for efficient transcription as they bind transcription factors)
- Enhancers and silencers, increase/ decrease the activity of a promoter.
How to measure transcription levels?
qRT-PCR, gel assay, Genome-wide approach (ChIP, limit is you need an antibody)
Transcription regulatory proteins
- DNA binding,
- prot-prot interactions,
- sites for signals (eg. GR),
- Some can cause chromatin change
Yeast GAL system
In the presence of galactose, GAL4 protein (trans element) is produced which activates transcription of other galgenes coding for enzymes by binding upstream enhancers(UAS). A key regulator is Gal4. Regulates expression of Gal1/2/7/10. Each of these genes has 2+ 17bp Gal4 binding sites (enchancers). Two critical domains, the DNA-bidning domain and the activation domain are needed for Gal4 to funciton. Gal80 normally blocks Gal4 function unless Gal3 inactivates it. Gal 3 inly binds to Gal80 when it is bound to galactose. Gal4 can both attract other proteins involved in initiating transcription and recruit proteins to modify chromatin structure.
PEPCK
- Binding of GR signal leads to chromatin remodeling (turn on gene by displacement of histories by acctyylation of histone tails).
- In the PEPCK example you saw a simple case where the binding of an activator (GR protein) helps recruitment of a chromatin remodelling complex. This is an example of cooperative binding and is typical in eukaryotes
Combinatorial control
- Transcription factors can be shared between target genes to be regulated. This is because they act in combination with others to differentially regulate the expression of the targets.
- The activity of a SOX protein is dependent upon the identity of its partner and the context of the DNA sequence to which it binds.
SOX-partners
- Bind to partners (other sox or other proteins. ).
- Flexibility in DNA motif recognition.
- spacing between DNA binding motifs
