Lecture 12 Transcription 2 Operons Flashcards
Operons
Common in bacteria rare in eukaryotes.
Groups of genes controlled by a shared promotor and all share a terminator. So RNA polymerase codes all genes in the group together.
Operator in the promotor binds proteins to tell RNA polymerase to start/not start transcription regulating genes on the operon by binding pos (activators) or neg (repressors) proteins.
Operons allows coordinated expression
1) catabolic - express genes to break down (e.g. enzymes lac operon)
2) biosynthetic - express genes to build (e.g. Tryp operon)
Can be controlled by pos/neg mechanism
Operons allow coordinate control of expression in prokaryotes:
- produce proteins bacteria needs at the same time
- all genes in operon under same control
- all switched on if needed/off if not
- depending on food availability
Food availability
Determines enzyme production e.g. lactose present: catabolic operon switches on lactose gene
How is food detected e.g. lactose operon
Lactose broken down into glucose and galactose by beta-galactosidase enzyme.
Three enzymes required.
Lactose permease opens a channel (pore) in cell wall of bacterium to allow in sugar.
Beta-galactosidase enzyme to split the molecule
Transacetylase attached acetyl groups to toxic byproducts (sometimes beta-galactosidase makes mistakes creating toxic sugar arrangements)
These 3 enzymes are coded by 3 genes on lac operon : lac z y and a
Transcription produces a polycistronic mRNA (mRNA thatc corresponds to multiple genes)
Regulatory gene LacI and functions of Lac I ZY and A
Codes for a regulatory protein that regulated Lac zya operon. Repressor binds to Lac operator so RNA polymerase cannot bind to promotor- blocking it aka ‘stearic hinderance’ - blocking the function of another protein
LacI- encodes repressor (tetramer)
LacZ- encodes enzyme beta galactosidase (tetramer)
LacY- encodes permease (membrane bound)
LacA- encodes enzyme transacetylase (dimer)
Inducer - allolactose
Binds to the 4 binding sites on the repressor tetramer making them inactive so they cannot bind. Inducer is present when lactose is present it is allolactose - a rearrangement of lactose produced when cell begins to break down lactose.
Then RNA polymerase can bind and transcription can occur.
Operon is never entirely on or off
It’s in equilibrium, essentially for function permease must always be present to allow lactose into cell and trigger cell response
Mutants used to explain how sugar levels control transcript levels
Useful to solve biological puzzles
Mutants that have an altered/defective genes may do things differently
By observing what is diff from norm we can identify genes necessary for bio processes
This is how Jacob and Manor gained proof of Lac operon:
Isolated and mapped E. Coli mutant strains that did not respond to lactose found mutations in the regions that define regulatory system of the lac operon.
They isolated two types of mutation: lacl- and lacOc.
They produced an operon model in 1969 to explain how sugar levels might control transcript levels and won 1965 Nobel prize
To do this they had to work out if genes were on or not. Detect expression of lacZ by adding lactose analogue xgal that forms a product that oxidises to form a blue precipitate in colonies expressing lacZ
As all operon genes are connected if lacZ is on all are on. Adding lactose to bacteria a visible blue response seen in 10 mins
In lacI- mutant the operon is never repressed. LacZ is constitutively (always) expressed in lacI- mutants so this shows that LacI would normally be a repressor
LacI+
Adding a LacI+ plasmid that overcomes lacI- proving lacI recessive
If lacI+ gene is present it doesn’t matter which bit of DNA lacI+ gene is on it will always restore normal function proved by:
Strain 1: LacI- lacZ+/F’LacI- LacZ-
Normal induction
Strain 2: LacI+ LacZ+/ F’LacI- LacZ-
This strain also shows norm induction
LacIs super repressed mutant
Has no inducer binding sites at all. Dominant and cannot be corrected
LacOc
Does not encode a protein it’s a DNA recognition sequence. LacOc constituent mutant not recognised by a normal repressor so operon never repressed. LacZ is constituently expressed - so LacO normally causes repression.
Strain 1
LacOc LacZ+/ F’ lacO+ lacZ-
Still constitutive extra wild type lacO doesn’t help - lacOc dominant
Strain 2
LacO+ lacZ+ / F’ lacOc lacZ-
Normal - mutation on plasmid cannot destroy normal functioning - LacO only affects genes it’s physically connected to - it’s a regulatory piec of DNA deciding if certain proteins bind or not
Lac O acts in cis - must be next to the thing it regulates on DNA
Summary
Lac operon comprised of 3 genes necessary for utilisation of lactose
Operon= coordinate control
LacI encodes repressor protein acts in trans
LacO defines site at which repressor protein acts - acts in cis
Repressor blocks access to promotor for RNA pol (stearic hinderance)
