Lecture 12 Flashcards
What is enzyme induction - why does it happen?
Enzyme induction is when bacteria only produce enzymes required for growth on a particular substrate in the presence of this molecule
- it happens cos bacteria are exposed to different environments (nutrients) and selection has led to the evolution of efficient systems
How do bacteria know when to produce the enzymes in enzyme induction ?
Regulation!
Positive and negative regulation
What is positive and negative regulation ?
Promoter: binds RNA polymerase and initiates transcription —> mRNA
Operator: binds regulatory proteins to alter transcription
In negative regulation: gene expression is turned off by a regulatory protein termed repressor
In positive regulation: gene expression is turned on by a regulatory protein termed activator (some promorotrs are weaker and need help to recognise?)
(- activator binds infront of the promoter (different spot to the repressor))
Inactive activator =
No expression
Inactive repressor=
Constitutive expression
How activators/repressors bind / unbind
- detect an effector
- undergo a conformational change
- this alters their ability to bind DNA
How us the lac operon regulated
The repressor protein controls the lac operon
What is an operon ?
MRNA that can give rise to many genes
How did they realise that inducers regulated new B-gal synthesis?
- originallly thought that inducers that lead to increased B-gal activated pre-existing enzyme
- but radioactive amino acids were added before or after induction, this resulted in radioactivity accumulating in B-gal enzyme
- new fast synthesis
- remove inducer and synthesis stopped
Are inducers different from substrates?
Yes
Lactose is an inducer (allolactose)
Lactose is a substrate
Other related molecules that are not substrates can act as inducers
Enzymes that break down lactose into galactose and glucose are not the same enzymes as the one that recognises the inducer
- this also shows that the component that recognises the inducer is distinct from the enzyme
How do we know genes are controlled together
- induction of permease led to co-induction of B-gal
- mutations in these genes showed that they were seperate genes
- gene mapping showed that lacZ,lacY and lacA are closely related
(This forms the idea of an operon and mRNA of them all together)
Has been verified experimentally
(This makes sence and permease breaks down lactose and B-gal brings it into the cell - no point bringing it in if u can’t break it down)
(Good cos one Operon can control a bunch of shit)
Key regulator in this pathway
LacI
Features of the Lacl repressor
- found mutations (lacl-) that were constitutive (always on) for production of enzymes
- did not respond to inducer - first regulatory mutant
- mapped close to lacZYA
(This told them there was a mutation close to the operon that could no longer respond to the inducer
The PaJaMo experiment - initial thinking
Initial thinking was that he L- allele makes an internal inducer (L- is always on)
Therefore L- will be dominant over L+ - WRONG
- when using gene transfer from Hfr to F- so that the bacteria have both genes, when no inducer is added, if F- was dominant it should keep producing B-gal but it doesn’t
Results of the PaJaMo experiment
- a diffusible repressor initially absent from the cytoplasm of the recipient cell
The PaJaMo experiment showed …
Making complememntation plamsids before cloning: formation of F-prime (F’) factors
- they are generated by excision by recombination with a distant IS element
- carry chromosomal DNA
- “in vivo” cloning vectors
- useful for doing complementation tests
- used before “modern” recombinant techniques
How its showed the at LACI+ is trans dominant over LacI-
(One operon on a plasmid and the other on in the chromosome)
This shows the lacI gene encodes a diffusable factor (repressor)
Features of lacIS
It is dominant and cannot bind inducer
- laclS mutants did not express lacZ, lacY or lacA
- mapped to lacl gene (super repressors)
- lacIS trans-dominant over lacl+ and lacl-
Inducer interacts directly with repressor
(Can’t respond to the presence of inducer - inducer would usually bind to repressor)
(Trans means it can move around the cell and have effects on other
Operator (LacI binding site) - Oc mutants features
- hypothesised repressor interacted with “operator” located near begining of genes it controlled
- searched for constitutive mutants in cell containing two copies of repressor (i.e hard for the repressor to mutate)
- operator-constitutive (Oc) mutants
(This also shows they operate on cis - their won genes not the adjacent)
What did the Oc mutants show?
- That the repressor interacts with the operator
- operator are cis-acting
- Oc cis-dominant over LacI+ and LacIs
- mapped betweeen lacI and lacZ
Negative regulation of the lac operon - the operon model
- repressor is produced
- sences the inducer (lactose)
- repressor binds to operator sequences if not bound to inducer
Key concepts
What is true for ecoli is true for the elephant
Kinda not really but kinda
Inducer vs substrate
Inducer binds to repressor
Substrate binds to enzymes that degrade it
How was the operon model duduced
- some biochemistry
- mutant isolation
- gene mapping
- complementation analysis (dominant or recessive alleles)
- tans vs cis effects
- deductive logic
