2) Control of Transcription Flashcards
Why is it important that the amount a gene is transcribed and translated be varied?
Not all proteins are required in the same amounts or at the same time
Switching transcription of genes on and off when needed SAVES ENERGY
E.coli: Only needs to transcribe genes needed to make proteins to use the food substrates
How do prokaryotes control the amount of transcription?
1) Cell can control which genes are transcribed (where in the DNA transcription starts and stops)
2) Can control the rate and amount of transcription
What are the 3 different types of control mechanisms used by bacteria? (Names Only)
1) Operons: Coordinate control of gene groups
2) Alternative sigma factors: Decides which genes are transcribed
3) Regulating transcription termination: Decides whether additional genes are transcribed
What are operons?
What are the 2 types?
Genes which have related function in bacteria are grouped together and are regulated in a coordinate fashion= Operon
Controlled by a shared promoter
Regulatory proteins bind to the DNA of the promoter and its operator to regulate expression
Example: 9 genes making the 9 enzymes which synthesise histidine= Operon
Can be controlled by positive or negative mechanisms
2 types:
1) Catabolic (e.g. lac operon)
2) Biosynthetic (e.g. Trp operon)
What is the difference between positive or negative control?
The DNA-binding protein that binds the promoter can be a positive or negative regulator
Positive= Transcription is switched ON
Negative= Transcription is switched OFF
What the difference between catabolic and biosynthetic operons?
Catabolic: When the operon is involved in breaking down substrates. Produce molecules they can use for extracting energy
Biosynthetic: Synthesising new molecules they require for life
Where are catabolic operons used?
Control expression of enzymes used in sugar metabolism and utilisation
What happens to catabolic operons when glucose is available?
Glucose= Preferred sugar
Do not needed to switch on expression of enzymes which break down other compounds and release sugars= Transcription= OFF, normally OFF
These operons (such as Lactose (lac) and arabinose (are) are ONLY switched on when their sugar is present
What does the lactose operon do?
Lac gene: Codes for the enzyme B-galactosidase which breaks down the sugar lactose= Cleaves the beta 1,4 linkage
Lactose: Disaccharide made up of 2 monosaccharide sugars joined together= Glucose + Galactose= E.coli can only use the monosaccharides
What are the different genes of the lac operon?
lacZ, lacY and lacA= Lac operon AND also lacI
Structural genes and are transcribed from a single promoter into polycistronic mRNA
What is the difference between structural and regulatory genes?
Structural genes: Code of enzymes or others that actually DO the work
Regulatory genes: Control the operon
What is the regulatory gene for the lac operon?
LacI= Gene that produces the regulatory protein. It has its own promoter and terminator
Product of lacI regulates expression of lacZYA
If its function is disrupted, all of the other 3 genes are expressed ALL the time
It encodes a REPRESSOR
What does lacZ do?
Encodes the enzyme beta-galactosidase which is very stable
Proof experiments: Always monitor lacZ as it is the easiest to detect= Only need to monitor one of them
Able to measure beta galactosidase activity easily= turns blue on X-gal
What does lacY do?
Encodes a permease= Transports lactose into the bacteria as it cannot diffuse through the plasma membrane by itself
What does lacA do?
Encodes an enzyme transacetylase= Add acetyl groups to toxic sugars= Makes them harmless
(Although no one really knows its function…)
How is the lac operon regulated when lactose is ABSENCE?
Also have: lacO= Next to lacZ, is the operator sequence= Site at which the repressor protein acts
1) lacI gene is transcribed= produces mRNA that gets translated to a protein
2) 4 copies of proteins aggregate together= Tetramer
3) IF THERE IS NO LACTOSE: tetramer can bind to the operator region (lacO)
4) Binding= Interferes with binding of RNA polymerase to the promoter by steric hinderance (gets in the way…)
5) Prevents the expression of operon= Transcription is switched OFF
Binding sites of RNA polymerase at the promotor and lac repressor OVERLAP= if protein binds= Gets in the way= RNA polymerase cannot bind
How is the lac operon regulated when lactose is PRESENT?
Prevents the repression mechanism from working
1) Always have some permease in the plasma membrane= Imports lactose
2) Beta-galactosidase can rearrange lactose= Produces side product allolactose= Inducer
3) Allolactose binds to lac repressor protein= Conformational change= inactive form that cannot bind to operator
4) RNA polymerase can bind to the promoter and transcribes the operon= Structural genes are transcribed and translated into enzymes
Allolactose is present whenever lactose is present
What does the lacI- mutant do?
lacI- mutant version of lacI gene= Produces version of the lac repressor protein which CANNOT BIND to the operator
Result: Expression of the 3 structural genes of lac operon are ALWAYS switched on= Transcription ON
lacZ gene is constitutively expressed= Their effect is to cause constitutive expression of lacZ and the other genes
How did Jacob and Monod discover the Operon model?
Took 2 strains of chromosomes:
F’= Extra inserted genes delivered on a small plasmid
+= Normal, effective wild type version
-= Defective mutant
1) lacI- lacZ+ / F’ lacI lacZ-
Chromosome has mutant lacI, F’ plasmid adds normal lacI joined to mutant lacZ
2) Lac+ lacZ+/ F’lac- lacZ-
Chromosome has normal versions of both genes, F’ plasmid adds mutant version os both
Asked question: Would extra copies of genes (on F’ plasmid) alter behaviour of original strain
What happened in strain 1?
Normal lacI gene on F’ plasmid makes normal version of repressor protein= Normal repression and induction
SHOWS: lac+ (wild type) is the DOMINANT as it can overcome the lac- mutation + the gene must encode a diffusible product that can move to the operator on the bacterial chromosome= MUST have the lac repressor protein
Strain 2: Also normal induction
What does it mean by “lacI acts in TRANS with respect to the lac operon’?
The lacI gene does not need to be on the same piece of DNA as the lac operon as there is a diffusible product that can move to the DNA when required= Lactose repressor
What do lacO^c mutations do?
Cis-dominant
LacO^c = cannot be recognised by a functional wild type repressor (lacI+)= Protein cannot bind= Operon is constitutively switched on
How did their cis-dominant property be discovered by Jacob and Monod?
Again took 2 strains:
1) lacOc lacZ+ / F’lacO+ lacZ-
Mutant and normal wild type lacZ is on bacterial chromosome, F’ plasmid adds normal wild type lacO operator with mutant lacZ
RESULT: Still constitutive, adding the extra wild type lacO does not help
(ii) lacO+ lacZ+ / F’lacOc lacZ-
Normal wild types on bacterial chromosome, and mutants on F’ plasmid added
RESULT: Normal, the 2 mutations on the plasmid do not destroy normal functioning
SHOWED: When lacO gene is on the SAME chromosome as the functional lacZ, it is dominant= Cis-dominant
LacO cannot produce a product that can diffuse and affect genes somewhere else
What does the lacI^s mutations do?
Super-repressed mutations and are un-inducible
RESULT: Make repressor insensitive to inducer= Mutant repressor will always be bound to the operator= Transcription is always switched OFF
