Regulation of transcription initiation Induction â lac operon Repression ...
Lecture Three (Tuesday, Sept 30) Looking ahead: Regulation of transcription initiation Induction – lac operon Repression – trp operon Stringent Response HOMEWORK TODAY – DUE NEXT TUESDAY I. Regulation - basic concepts constitutive expression = no control by environmental conditions. Always on. Guess some genes that would be constitutive.
negative control = gene is usually expressed; turned off by a repressor protein positive control = gene is usually off; turned on by an activator protein repression = synthesis of a gene product is turned off by the end product, the corepressor. Classic example: trp operon induction = synthesis of a gene product is turned on by appearance of a substrate, the inducer. Classic example: lac operon
operon = a set of related genes controlled as a single genetic unit operator = DNA binding site for a repressor or an activator
II. Induction : Lac Operon - general Thought Question (from Friday): Say you don’t need a gene very often. How would you keep it off? (But are able to transcribe it at high levels when you want it)?
• Overall function of the operon is to break down lactose into galactose + glucose for energy • catches: 1. E. coli isn’t always sitting in a puddle of milk. 2. Even if E. coli is in a puddle of milk, it would prefer to eat any glucose first. Ë operon is only ON when you have lac but don’t have glc • How is this accopmlished? Two main functions need to be regulated. 1. Enzyme that breaks down lactose into gal + glc (b-galactosidase) 2. Enzyme that transports lactose into the cell - lactose doesn’t diffuse in at all. (permease activity) • Parts: Three structural genes: lacZ = b-galactosidase lacY = lactose permease lacA = transfers an acetyl group from Acetyl-CoA to b-gal One regulator = lacI = repressor can bind to operator binds to inverted repeats (dyad symmetry) binds as a dimer ** these features are common with regulatory DNA binding proteins
ONE promoter (yields a polycystronic mRNA) ONE operator = region of DNA near promoter that determines activity of the promoter
III. Regulation
of the lac operon
A, No lactose present: operon is off • LacI is bound to the operator Here the operator and promoter overlap. • LacI holds RNAP at the promoter • prevents an open binary complex from forming • “nothing” is being made B. Lactose becomes available: operon is expressed • lactose binds to LacI and changes its conformation; LacI falls off of the operator. • allosteric control = change (such as binding a ligand) at one site affects a distant site in the protein. Here: binding inducer affects the DNA binding site. • promoter is now available for RNAP to bind and transcribe lacZYA transcript. Both transcripts are translated efficiently. • LacY transports lactose into the cell • LacZ (b-galactosidase) breaks down lactose, cell eats happily
What are we missing?
FIGURES: Dyad symmetry in binding site (Fig 10-12) homodimer LacI binding structures of IPTG, X-gal • Jacob and Monod studied the lac operon because it is easy to determine if the operon is on/off. To do this, they used the lac analogs IPTG and X-gal. IPTG functions as the inducer molecule X-gal is cleaved by b-galactosidase: X-gal (colorless) ‡ X (blue) + galactose • Result: operon is ON = blue colony; operon is OFF = white colony
C. Cell eats all of the lactose Based on what I’ve told you so far talk to your neighbor and make a guess how this situation would work.
IV. lac mutants ** study of mutants of the system was vital to understanding the operon
lacI• repressor has a mutation so that it cannot bind to the operator • no way to shut off the system (irregardless of lactose) Ë What if you supply a wild-type copy of lacI? LacI can diffuse within the cell and bind to the operator. Ë
lacI- mutations are recessive. LacI is trans-acting.
lacOc • operator has a mutation that prohibits the repressor from binding • no way to shut off the system (irregardless of lactose) Ë What if you supply a wild-type copy of lacO? there isn’t a diffusible product = cis-acting mutation mutant operon still will not function this type of mutation is dominant Ë if you supplied a wild-type operon to the cell, the phenotype of the cell would be Lac+ (normal regulation) but this mutant operon will still be defunct
lacIs • repressor cannot bind inducer (but can still bind to DNA) • repressor will always be bound to the operator Ë What if you supply a wild-type copy of lacI? Ë
placpromoter has a mutation so that it is non-functional Ë What if you supply a wild-type copy of plac? a. Without a functional operon behind it? Ë
b. With a functional operon behind it? Ë
Francois Jacob
Jacques Monod
Winners of the Nobel Prize in 1965 for their work on the lac operon.
Dyad symmetry in the LacI binding site
Transcription start site
Fig. 10-12
1
LacI binds as a homodimer
(this is a common theme among DNA-binding proteins)
Transcription start site
Fig. 10-12
Not in text
2
negative control = gene is usually expressed; turned off by a repressor protein positive control = gene is usually off; turned on by an activator protein repression = synthesis of a gene product is turned off by the end product, the corepressor. Classic example: trp operon induction = synthesis of a gene product is turned on by appearance of a substrate, the inducer. Classic example: lac operon
operon = a set of related genes controlled as a single genetic unit operator = DNA binding site for a repressor or an activator
II. Induction : Lac Operon - general Thought Question (from Friday): Say you don’t need a gene very often. How would you keep it off? (But are able to transcribe it at high levels when you want it)?
• Overall function of the operon is to break down lactose into galactose + glucose for energy • catches: 1. E. coli isn’t always sitting in a puddle of milk. 2. Even if E. coli is in a puddle of milk, it would prefer to eat any glucose first. Ë operon is only ON when you have lac but don’t have glc • How is this accopmlished? Two main functions need to be regulated. 1. Enzyme that breaks down lactose into gal + glc (b-galactosidase) 2. Enzyme that transports lactose into the cell - lactose doesn’t diffuse in at all. (permease activity) • Parts: Three structural genes: lacZ = b-galactosidase lacY = lactose permease lacA = transfers an acetyl group from Acetyl-CoA to b-gal One regulator = lacI = repressor can bind to operator binds to inverted repeats (dyad symmetry) binds as a dimer ** these features are common with regulatory DNA binding proteins
ONE promoter (yields a polycystronic mRNA) ONE operator = region of DNA near promoter that determines activity of the promoter
III. Regulation
of the lac operon
A, No lactose present: operon is off • LacI is bound to the operator Here the operator and promoter overlap. • LacI holds RNAP at the promoter • prevents an open binary complex from forming • “nothing” is being made B. Lactose becomes available: operon is expressed • lactose binds to LacI and changes its conformation; LacI falls off of the operator. • allosteric control = change (such as binding a ligand) at one site affects a distant site in the protein. Here: binding inducer affects the DNA binding site. • promoter is now available for RNAP to bind and transcribe lacZYA transcript. Both transcripts are translated efficiently. • LacY transports lactose into the cell • LacZ (b-galactosidase) breaks down lactose, cell eats happily
What are we missing?
FIGURES: Dyad symmetry in binding site (Fig 10-12) homodimer LacI binding structures of IPTG, X-gal • Jacob and Monod studied the lac operon because it is easy to determine if the operon is on/off. To do this, they used the lac analogs IPTG and X-gal. IPTG functions as the inducer molecule X-gal is cleaved by b-galactosidase: X-gal (colorless) ‡ X (blue) + galactose • Result: operon is ON = blue colony; operon is OFF = white colony
C. Cell eats all of the lactose Based on what I’ve told you so far talk to your neighbor and make a guess how this situation would work.
IV. lac mutants ** study of mutants of the system was vital to understanding the operon
lacI• repressor has a mutation so that it cannot bind to the operator • no way to shut off the system (irregardless of lactose) Ë What if you supply a wild-type copy of lacI? LacI can diffuse within the cell and bind to the operator. Ë
lacI- mutations are recessive. LacI is trans-acting.
lacOc • operator has a mutation that prohibits the repressor from binding • no way to shut off the system (irregardless of lactose) Ë What if you supply a wild-type copy of lacO? there isn’t a diffusible product = cis-acting mutation mutant operon still will not function this type of mutation is dominant Ë if you supplied a wild-type operon to the cell, the phenotype of the cell would be Lac+ (normal regulation) but this mutant operon will still be defunct
lacIs • repressor cannot bind inducer (but can still bind to DNA) • repressor will always be bound to the operator Ë What if you supply a wild-type copy of lacI? Ë
placpromoter has a mutation so that it is non-functional Ë What if you supply a wild-type copy of plac? a. Without a functional operon behind it? Ë
b. With a functional operon behind it? Ë
Francois Jacob
Jacques Monod
Winners of the Nobel Prize in 1965 for their work on the lac operon.
Dyad symmetry in the LacI binding site
Transcription start site
Fig. 10-12
1
LacI binds as a homodimer
(this is a common theme among DNA-binding proteins)
Transcription start site
Fig. 10-12
Not in text
2
