The Operon Flashcards
hairpin helix hairpin
part of the alpha subunit
- makes contact with the minor groove
- there are 2 HHH domains
what groove of the DNA interacts with alpha CTD?
- the minor groove
- the AT richness of the UP site contributes to the narrowness of the minor groove where HHH makes contact
how does the binding of the alpha CTD to the UP site stimulate transcriptional activity?
- the alpha subunit of the RNA polymerase is a determinate of promoter strength
- C terminal domain of alpha binds binds upstream to a UP site from -40 to -60
- the UP element of 2 subsites (proximal and distal) - you need two sites in the UP to support both alpha CTD units
- each site is occupied by an alpha CTD with the proximal site being the strongest
- the alpha subunit has affinity for the UP element site and increases RNA pol binding to the area
- increases polymerase binding
how can the binding of alpha to the UP sometimes stop transcription?
- UP element determined by binding selection technique
- UP elements often increase the recruitment of RNA pol to the promoter
- sometimes there is no effect but can have a negative effect by preventing efficient promoter clearance
what is the overall base composition of the UP?
UP site is AT rich
at what stage of transcription does the alpha CTD have the most influence?
during the formation of the closed promoter complex
what principle mechanism did the experiment with the substitution of the CTD of the lambda repressor with the alpha subunit demonstrate?
- result: tethering to the promoter of a protein capable of interacting with any subunit of RNA pol will activate transcription
- conclusion: contact with the CTD effects recruitment but probably not initiation or enzyme activity of RNA pol
- example of an artificial activator
what is the strongest promoter in bacteria?
the UP element
CAP activator
- provides contact for the CTD, which alpha increases affinity for
- alpha regulates when CAP binds
operon definition
- a complete regulatory system which includes the structural genes encoding proteins or RNAs, and the promoters and cis elements
- does NOT include the genes encoding specific regulatory proteins such as activators and repressors, these are separate from the operon
cis elements
DNA sequences that regulate transcription by binding to RNA pol or regulatory proteins
trans elements
proteins that regulate transcription by binding to cis-elements
- not restricted to one promoter
operator
- site for repressor binding
- if a repressor is involved in activation, the cis activator is the operon
polycistronic mRNA
- messenger RNA that encodes more than 2 proteins
positive regulation
- promoter is deficient, need an activator to bind to the site for gene expression
negative regulation
promoter will be active and a repressor binds to an operator to shut down gene expression
- gene is on by default
how do you activate if under repression?
- induction by a de-repressor
how do you repress if you have an activator?
- change the conformation of the activator so it doesn’t bind
what types of trans-activators are associated with positive and negative regulation?
- repressors
- activators
- bind to the cis-elements
what general mechanism of repression applies to most bacterial repressor proteins? Does this mechanism work for eukaryotes?
- the repressor protein will sit on the DNA where RNA polymerase binds
- essentially gets in the way and prevents binding
- this mechanism does NOT work for eukaryotes because their DNA is under transcription all the time
- their regulation is through histones, which is the consequence of a large genome
induction under negative control
- requires an inducer
- when under repression, the repressor is bound to the DNA and stops transcription
- when there is an inducer, this changes the conformation of the repressor and allows for gene expression
induction under positive control
- requires an inducer
- when under repression it has an inactive activator, so no gene expressed
- when induced, an inducer binds to the activator and causes a conformational change for gene expression
repression under negative control
- requires a corepressor
- in induction, the repressor is inactive and the gene is expressed
- under repression, the repressor binds with a corepressor and causes a change in conformation, which stops the gene from being expressed
repression under positive control
- requires a corepressor
- in induction, the activator is active and causes gene expression
- under repression, the corepressor binds to the activator and causes it to become inactive and there is no gene expression
what are the two reasons there are so few lac repressors in e. coli?
- weak promoter (low match to sigma 70)
- very poor translation due to short 5’ UTR - there is no SD and starts with GUG
what is the role of Beta-galactosides in the lac operon?
- small molecules that induce an operon fir a catabolic pathway are identical with or related to the substrate for its enzymes
- B-gal are the substrates for the enzymes encoded by lacZYA
- in the absence of B-gal, it is poorly expressed
- B-gal cleaves lactose to form allolactose, which is the signal to start transcription
- however, lac mRNA is unstable and can be reversed
what two binding sites are present on the lac repressor?
- binding domains for the operator and inducer
- repressor inactivated by allosteric interaction
what would the transcription state be if mutations occurred in the operator?
- cause constitutive expression of all 3 lac genes
- cis-acting mutants only have to be mutated once to not work
- means the transcriptional state would always be on and no longer be regulated
what would be the transcriptional state of the lac operon be if mutations occurred in the DNA binding domain of the repressor?
constitutive because they can’t bind to the operator
what would the transcriptional state of the lac operon be if mutations occurred in the IPTG binding site?
- prevent it from being inactivated
- causes inducibility
explain why the lacI-d mutation is dominant
- lac repressor is a tetramer of identical subunits
- if you put a mutation in one of the monomer, the whole subunit will be effected, causing no repression
- occur in the DNA-binding domain
what is the evidence the inducer molecule binds the lac repressor when the repressor is still bound to the operator site instead of binding to the free repressor?
- inducer binds directly to release from the operator
- in vitro IPTG causes immediate release from repression
- half life of repressor DNA is 15 minutes
How can a DNA binding domain that only recognizes one face of the DNA helix be used to regulate a specific gene since the 5-6 nucleotides of the binding site would occur frequently in the genome by serendipity? (Hint: palindrome, dimer)
- the lac operator has a 26 bp symmetrical sequence (palindrome)
- binds to the repressor as a dimer
- each inverted repeat binds a repressor subunit
- has a dimer that reads both sides of DNA
write the equation for the association constant of a repressor for DNA
Ka = [ repressor-DNA] / [free repressor][DNA]
- non-specific equilibrium binding constant = 2x10-6 M-1
- non-specific binding site concentration = 7x10-3 M
- .01% of the lac repressor is bound to DNA
What is the Ka for lac repressor binding to operator DNA (no inducer present)?
x
What is the Ka for lac repressor binding to non-operator DNA (no inducer present)?
x
What is the Ka for lac repressor binding to operator DNA in presence of inducer?
x
What is the Ka for lac repressor binding to non-operator DNA in presence of inducer?
x
what two mechanism is the lac operon under?
positive and negative control
what is not prevented from binding in the lac operon?
- the RNA polymerase is not prevented from binding
- RNA polymerase and repressor have affinity for one another
- The lac repressor binds underneath the RNA polymerase
- RNA polymerase is 100 fold greater affinity for the lac promoter if the repressor is already there
- This prepositions the RNA polymerase so that when it sees lactose it can immediately activate and use the lactose
what is the make-up of the lac operators?
- two always have to be simulataneously occupied by the lac repressor
- two dimers are tied together by a leucine zipper and each monomer contains a component of the zipper
- four membered leucine zipper at the C terminus of the lac repressor
- represses by clamping on the DNA so sigma cannot melt the strands
what are the lac genes controlled by?
- transcription of the lacZYA gene cluster is controlled by a repressor protein
- the repressor protein is a tetramer of identical subunits coded by the gene lacI
- expression of lacI is very low at only 10 repressors per cell
catabolism
the systems that allow substrates to induce operons coding for metabolic enzymes
biosynthesis
end-products repress the operons that code for biosynthetic enzymes
what are repressors and what do they bind to?
transcriptional control proteins that down-regulate transcription by a variety of means. The most common is to block access of RNA polymerase to the promoter by binding to, or near, core elements. In many cases the same protein can act both as a repressor and as an activator, depending on the context.
- Repressors bind to operator sites.
what are mutations in the lacI gene?
- trans-acting
- affect expression of all lacZYA clusters in the bacterium
- mutations that totally eliminate lacI function are constitutive
what is the main determinate of repressors?
- location
- single repressor may control multiple promoters
- different mechanisms of repression are used in different positions of the repressor
- repressors can bind to several different locations with respect to the promoter
What is meant by the specificity of binding? What is it for the Lac repressor with and without inducer?
- the size of the genome dilutes the ability of a protein to bind specific target sites
- The specificity of the protein counters the effect of the mass of the genome (DNA ) effect. Therefore, the amount of protein needed increases with the size of the genome and decreases with binding specificity.
hinge helix (helix 4) domain involved in monomer-monomer binding
globular domain
hinge helix domain involved in joining dimers into a tetramer?
leucine zipper at the C terminus
inducer binding pocket
located at the core
what feature of the lac repressor binds to the minor groove? the major groove?
- hinge helix 4
- HTH
when is helix 4 of the repressor not an alpha helix?
when it leaves the minor groove and becomes floppy
what is another name for a triple-helical cluster reinforced by b-strands?
helix-turn-helix
When the inducer binds to the Lac repressor tetramer, what causes it to decrease affinity for the operator site? (mechanism) (Hint: scissors motion) Do the two dimers separate, or do the two monomers that make a dimer separate?
Two DNA binding domains will bind and cross each other
The DNA binding domains will rotate and separate when IPTG binds
Too far apart for both to bind to the operator at the same time
Causes loss in affinity
No cooperativity
Destroys repression
How many operators for the Lac repressor are present in the E. coli genome?
two
what does the DNA-binding domain contain?
a HTH motif (3) and a hinge helix
what is the headpiece of the lac repressor?
- N-terminus is called the headpiece and contains a helix-turn-helix domain
where does the inducer bind in the lac repressor?
cleft between the two core domains
what does the C-terminus contain?
- an alpha-helix with two 7aa repeats (leucine zipper)
what occurs when the lac repressor changes conformation?
Inducer changes the structure of the core so that the headpieces of a repressor dimer are no longer in an orientation that permits binding to DNA.
what happens to the lac repressor when it binds to the inducer molecule?
the two monomers of the dimer separate so that only one at a time can bind to the operator
Is RNA polymerase More, or Less, likely to bind to the lac promoter when the repressor is present?
more
- The presence of the Lac repressor at the promoter increases the affinity of RNA polymerase for the lac promoter by 100-fold.
When RNA polymerase and the Lac repressor are both bound to the lac promoter, what keeps transcription turned OFF?
- dependent on CAP
- when the repressor leaves, another protein needs to bind in order to activate gene expression
what is occurring in the media is a DNA loop in present?
the gene is inactive
The Lac repressor contains a 4-helical leucine zipper. What is meant by the term “hydrophobic heptad repeat”?
x
how does the lac repressor inhibit transcription?
inhibits the transition from the closed to open promoter complex
In the abcdefg repeat structure of a leucine zipper, which positions contain bulky hydrophobic residues? At which position is a leucine often found?
leucine is found at positions a ad d
Why is a leucine zipper sometimes called a “coiled coil”?
Oligomeric transcription factors are often held together through a-helical regions
Describe how the lambda phage repressor can act both as a repressor and as an activator protein.
LexA undergoes autoproteoalysis by RecA
Lamba mimics LexA
RecA targets lamba to cleave for derepression
Lysis the cell
When the cell is under attack, this is when lamba will leave and RecA is activated
Operator L makes contact with RNA polymerase
This example is both repressor and activator
How many operator sites are present in the araBAD operon? How many total sites for AraC binding?
- Catabolite-repression operon (like lac).
- CAP site is 200 bp upstream; positive regulator
- AraC binds arabinose and changes configuration
to then bind araCI1and I2 sites.
Why are some of the AraC binding sites called “Inducer sites”?
x
What is the function of the DNA loop at the araBAD promoter?
operators must be on the same helical face.
When AraC regulates its own synthesis (autofeedback), what is the mechanism of repression?
araC is acting like a classical repressor in blocking RNA polymerase binding.
