1.) bind DNA directly, or are recruited to DNA by other factors
2.) Influence transcription (positively or negatively), either by directly recruiting polymerase (or blocking it), or indirectly by recruiting other factors
Cis-acting elements (DNA) (5)
promoters
enhancers
regulatory binding site
regulatory sites
activator/repressor sites
Sequences on the DNA that are recognized and bound to by trans-factors... (2)
Can contain a single binding site, or a cluster of sites (e.g. enhancers)
Can be near the start site (i.e. promoters) or far away (e.g. enhancers)
Transcriptional regulation can occur in __ or ___
cis or trans
You create a mutant cell line that is unable to express Gene X.
How do you determine whether the mutation is in the promoter (or other
cis-acting element) for Gene X, or in a trans-acting factor that positively
regulates transcription of Gene X?
Introduce a 2nd copy of Gene X and see whether its expression is affected by the mutation
Mutation in the Cis-acting element
Regulation in cis occurs when a region only affects transcription of the DNA strand it is on (i.e. it cannot impact transcription of another DNA strand). Regulatory sites on the DNA act in cis.
Mutation in the Trans-acting factor
Regulation in trans occurs when a factor can affect transcription on any DNA strand. Transcription factors act in trans.
You create a mutant cell line with no expression of Gene X. You introduce a second copy of Gene X in a plasmid (complete with Gene X’s promoter). The second copy of Gene X is expressed. Is the mutation in
A.) a cis-acting element
B.) a trans-acting factor
A.)
You run a Northern blot and probe with Gene X (will detect both
copies). If the mutation was in a trans-acting factor, which one would your blot look like?
B.)
Trans-acting factors bind to..... (5)
transcription factors (repressors and activators)
architectural regulators
cofactors (coactivators and corepressors)
insulator-binding proteins
effectors
cis-acting elements bind to.... (3)
regularoty sites (enhancers, promoters, etc.)
Architectural regulator-binding sites
insulators
Transctiption factors: repressors...
negatively regulate transcription by competing with RNA pol or other activators for binding to the promoter
Transcription Factors: Activators ....
positively regulate transcription by recruiting or enhancing recruitment of the transcription machinery to the promoter
Describe how transcription factors can act from long distances (eukaryotic)
Transcription activators bind to distant regions from the promoters (i.e. enhancers or regulatory sites) and help to recruit transcription machinery to promoters. Enhancers can be very, very far away from the promoter, like megabases
causes DNA looping
DNA looping (eukaryotic)
When a transcription factor bound to an enhancer binds to the promoter, it links the two distant regions and forms a loop of DNA between them
Architectural Regulators (eukaryotic)
Architectural regulators bind to DNA at architectural regulatorbinding sites and alter the structure of the DNA/chromatin, which indirectly facilitates transcription by making it easier for distant transcriptional activators to reach the promoter.
Cofactors
Cofactors (coactivators and corepressors) are proteins that do not bind DNA directly, but can facilitate interactions that activate or repress transcription
Insulators (eukaryotic)
Insulators are cis elements that block activators and repressors from being able to affect transcription from one side of the insulator to the other.
Also called boundary elements.
How they do this is still unclear. They probably block DNA looping.
Effectors
Effectors are small molecules that can bind to transcriptional regulators and affect their function.
• Effectors (as a class) can help to repress transcription (inhibitor), or they can help to activate transcription (inducers)
• Effectors are sometimes metabolites produced by the gene they regulate, and can act in a positive or negative feedback loop
Which trans-acting factor both binds to a cis-acting element as well as directly recruits RNA polymerase?
D. )
Proteins that act by bridging activators and RNA polymerase are called:
D. )
The lac Operon (image)
lacZ, lacY, and lacA –
encode proteins that metabolize lactose
lacI and lacO -
regulate when lac operon is expressed
Mutations in lacI or lacO lead to uncontrollable expression of....
Lac genes
lacI and lacO are important for ______ lac operon expression
repressing
lacI
encodes protein called lac repressor
lacO
binding site for lac repressor (called an Operator)
Lac repressor binds to the operator sequence to block....
Lac Expression
Does lacO act in cis or in trans?
A. Cis
B. Trans
A.)
Does lacI act in cis or in trans?
A. Cis
B. Trans
B.)
In the absence of lactose, .....
the lac repressor blocks transcription
In the presence of lactose, .....
the lac repressor is blocked, allowing transcription
An inducer (a type of effector)
binds to the lac repressor, .....
blocking its function and allowing lac expression
It’s called an inducer because it induces the operon, not because it induces the repressor (i.e. the inhibitor of an inhibitor is an activator)
The inducer is called allolactose
Allolactose
allolactose is a byproduct of lactose metabolism by the lac operon
when allolactose is produced, it blocks the lac repressor allowing lac expression
What a minute?! You need the lac genes
to be turned on in order to produce
allolactose, but allolactose is required to
induce the lac genes. So how do you turn
on the lac genes if your inducer requires
them to already be on?!!!
The lac repressor isn’t 100% efficient, and a low basal level of the lac genes are always expressed, producing just enough allolactose when lactose is present to block the lac repressor.
There are actually 3 lac
repressor binding sites on the lac operon:
O1, O2, and O3
O_ is the original and is the only one required
1
The lac repressor binds to O1 and either O2 or O3, creating
a loop
The loop physically blocks ___ _______
RNA polymerase
What would happen if you mutated lacZ?
A. You wouldn’t be able to metabolize lactose, but the
other Lac genes would still be expressed
B. The lac operon would become constitutively expressed
C. You couldn’t make allolactose, and couldn’t stop the lac
repressor
D. The same effect as if you mutated lacI or lacO
C.)
Lactose is not as efficient an energy source as ....
glucose
How does the lac operon know when glucose is present?
Cyclic AMP (cAMP) is over-produced when glucose is absent, but turned off when glucose is abundant
When glucose is not there, cAMP ....
is how the cell tells Lac operon and other sugar metabolizing operons that it’s time to turn on. cAMP is an inducer of Lac expression.
cAMP binds to a protein called _______ to
activate it
cAMP responsive protein (CRP)
CRP-cAMP binds to the Lac promoter to activate transcription (i.e. it’s an activator)
In the absence of lactose, the lac repressor is still bound, preventing CRP cAMP from turning on the lac genes when there’s nothing for them to metabolize.
glucose high, cAMP low, lactose _____
absent
In the presence of glucose, there’s no cAMP-CRP, so no effect on the lac operon.
no gene expression
glucose low, cAMP high, lactose ____
absent
In the absence of glucose, cAMP-CRP can bind to the Lac promoter, but if lactose is also absent, the lac repressor blocks cAMP-CRP
no gene expression
glucose high, cAMP low, lactose ____
present
When glucose is high, there’s no free cAMP-CMP. If lactose is also present, the lac repressor is blocked, but the positive signal given by cAMP-CMP is not there to help and only very low transcription can occur.
low level of gene expression
glucose low, cAMP high, lactose ______
present
When glucose is low, cAMP-CMP bind to the lac promoter. If lactose is high, then the lac repressor is unbound and high transcription
high level of gene expression
X-gal
a substrate for lacZ (b-galactosidase)
• Turns blue when lacZ is active
• Used to measure lacZ expression
IPTG
an inducer, binds to lac repressor to block it, allowing lac expression
researchers wanted to study mutations in the lac operon. They treated their mutants with IPTG to activate the operon, then added X-gal to visualize lacZ expression. In their mutant, the cells did not turn blue, indicating a problem with ....
lac expression
Which of the following statements about regulation of the lac operon is true?
A. When glucose is present, gene expression is activated by lactose.
B. The amount of glucose in the growth medium does not affect the
level of gene expression activated by lactose.
C. The binding of the Lac repressor to the operator is not affected by
the presence of glucose.
D. Gene expression is high when the Lac repressor is not bound to the
operator and glucose is available.
C.)
You are studying the lac operon, and want to switch on expression
of the lac operon, by adding something to your media. Which of the following would not work to turn on the lac operon? (assume there’s no glucose in your media)
D. )
How do proteins recognize specific DNA sequences?
Regulatory binding site sequences are often inverted repeats
Why?
because DNA binding proteins are often dimers
Helix-turn-helix motif: Recognition helix
one helix, fits into major groove
Most DNA-binding motifs
recognize the ____ groove, because it has more features to interact with (hydrogen bond donors/acceptors, etc.) than the minor groove
major
Another helix-turn-helix motif: Homeodomain
Dimer of helix-turn-helix protein
Each dimer contains one helix that binds to DNA
Leucine Zipper
A motif consisting of several leucines spaced about 7 amino acids apart
Zipper regions hold two subunits together, not involved in DNA binding, but are linked to a DNA binding region
not part of DNA recognition but involved in dimerization
Zinc Fingers
Zinc finger motifs are held together by Zinc ions. Each motif has a DNA recognition helix. Each motif is weak, but can be stacked together (beyond dimerizing), with each motif acting like a finger, gripping the DNA by the major groove.
Zinc ion stabilizes structure, but is not directly involved in DNA recognition
Transactivation domains
Transactivation domains are diverse and don’t share common motifs
These domains are on separate regions of the protein, allowing researchers to separate them (and swap them). This demonstrates the modular nature of regulatory proteins.
Transactivation domains have 2 basic domains:
1) DNA binding domain
2) Transcription-activation domain
Which sequence is an inverted repeat?
C. )
What region of each of the 3 major motifs actually recognizes the DNA?
E. )
You synthesize a fusion protein with JEL4 and RexA. You create a reporter construct to express lacZ, but containing the promoter for JEL4 and the binding site for RexA. Which domains should you use for JEL4 and RexA fusion protein?
A. Transactivation domain for JEL4, DNA-binding domain for RexA
B. Transactivation domain for RexA, DNA-binding domain for JEL438
