General Bacterial transcriptional regulation
Negative regulation of the lac operon
Negative regulation of the lac operon
Positive Regulation of the lac operon
100
Define negative vs positive regulation
positive- Increasing transcription rate under certain conditions
Negative- Decreasing transcription rate under certain conditions
100
For the negative regulation of the lac operon, the lac operon is controlled by an ____ that is subject to regulation by a the binding of a _____
operator
repressor/inducer
100
What do we mean by operators are cis- acting?
Cis- means on the same DNA molecule
so, cis acting means that an operator can only control the expression of genes that are on the same DNA, physically next to it
100
For positive regulation of lac operon, we have an lac operon activator which is called
CAP (catabolite activator protein)
200
What is an activator protein vs a repressor and how do each do their job.
Activator protein binds to the binding site and and helps RNA polymerase II start doing its job
Repressor proteins on the other hand prevent transcription, by attaching to the operator.
200
Why does lactose have to be present in order for the lac operon to be transcribed?
When lactose is present, it is converted into allolactose, which acts as an inducer molecule that binds to the allosteric site of the repressor, causing repressor to change shape, and detach from the operator, so now that the repressor is no the in the way, transcription can occur
**** Effector is a broad term for any molecule that can bind to a repressor or activator, but a inducer is specifically a type of effector that binds to the repressor protein
200
What is the F plasmid that we put into E coli?
Experiemental method where the F plasmid is a circular piece of DNA we put into E coli (bacteria), so this new plasmid and original bacterial DNA can exchange genetic material.
200
What binds to the allosteric domain of the activator CAP
cAMP
300
What is the operator vs the activator binding site
operator is the DNA site downstream of the promoter, where repressor protein binds to inhibit transcription
Activator binding site is the DNA site upstream of the promoter where an activator protein can bind and activate transcription
300
Describe what each letter on the LAC OPERON stands for
P - Promoter: Where rna polymerase binds to start transcription (lacP)
O- Operator: DNA sequence where the lac repressor protein binds (lacO)
Z- lacZ Gene: encodes the protein β-galactosidase (lacZ)
Y - LacY Gene: encodes the protein Permease (lacY)
A - LacA Gene: Encodes protein Transacetylase (lacA
300
What do we mean by operators are cis- acting?
Cis- means on the same DNA molecule
so, cis acting means that an operator can only control the expression of genes that are on the same DNA, physically next to it
Can be demonstrated with E coli and F plasmid experimentation. We inject a the F plasmid into the E coli. In the original E coli DNA, transcription is blocked because repressor is doing its job (O+ means operator is normal)
However, in the F plasmid, repressor cannot do its job because operator is mutated (Oc) so transcription goes on and on when it is not supposed to.
However, operators are CIS acting so that mutated operator in plasmid has no effect on the original DNA because it only affects its own plasmid, it doesnt transfer over
300
How do glucose levels regulate cAMP levels, and how does cAMP affect CAP
When glucose is high, cAMP is not produced
When glucose is low, cAMP is made, which then goes on to bind to the allosteric site of the activator CAP, which is return helps CAP turn on transcription, as it allows the CAP to connect with the RNA Polymerase II
400
what does the allosteric domain of the repressor and the activator protein do?
A site in the repressor where a allosteric molecule (AKA the effector) comes and binds to, when it binds it changes the change of the repressor in a way that causes it to detach from the repressor causing transcription to occur now that the repressor is not in the way.
In the activator, there is also a site where the effector binds, causing the activator protein to shape change where it can start activating transcription.
400
If no lactose is present, what occurs?
the repressor is still stuck onto the operator, so transcription is blocked.
400
Now, what do we mean by repressors are trans acting?
If we have E coli with a mutated lacI gene ( the gene that encodes the lac repressor protein), so we have a mutated repressor protein, so now it cannot bind to that operator so now trancription is constantly on
however, if we inject new plasmid (F) with normal LacI gene, repressor protein is normal, and can bind to operator and block transcription. the effect can TRANSFER OVER to the original DNA, so now this E coli that initially had a problem of constant trancription can be regulated and stopped appropiately because the effect of the F plasmid transferred over.
400
What happens if you have glucose present, lactose absent?
no lac mRNA
Glucose present means no cAMP, so the activator CAP will not bind to activator binding site, so transcription cannot be turned on
and no lactose means the repressor will stay stuck on the operator, so another reason for transcription to not turn on.
500
What is an operon and why do bacteria have operons?
An operon is a cluster of many genes that is controlled by a single promoter and regulatory sequence (by regulatory sequence I mean operators where repressors bind and activator binding sites where activators bind)
Bacteria are much simpler and have limited energy, thus operons are more effecient in producing multiple proteins all that the same time
500
What happens if there is a mutation in the operator sequence? And how do we label that
It prevents the repressor from binding, so lac operon is always on even in the absence of lactose, so transcription is constantly occuring and cant be stopped
Oc - meaning operator constitutive
500
what are super repressors?and what is the symbol for it
A super repressor is a different type of mutated form of the lac repressor protein that now causes the repressor to bind constantly to the operator, even when lactose is present, so as a result we have transcription inconveniently stopped. (symbol for super repressor is Is)
They are dominant so, even if we inject F plasmid with normal lacI gene encoding normal repressor, nothing will happen because super repressors are dominant
500
What happens if no glucose is there and lactose is present?
lots of lac mRNA being made
no glucose means cAMP is present, so cAMP can bind to CAP allowing CAP then to bind to the activator sequence allowing for transcription to be on
the effect is further amplified by lactose being present, it gets rid of the repressor protein on the operatoe, so now we have so much mRNA being transcribed
600
what does an inducer do to a repressor protein?
An inducer molecule binds to a repressor protein, causing a conformational change (a change in its shape) that prevents the repressor from binding to its DNA target, the operator
600
Now, what happens if there is a mutation in the repressor protein?
Mutation occured in the gene coding for this repressor protein, what is that gene called?
Mutated repressor protein is not functioning so it cannot bind to operator, causing transcription to occur constantly even in absence of lactose.
The gene is called lacI
I is the denoted letter for the gene downstream of the lac operon that codes for that lac repressor protein that goes on to act on that operator of the lac operon later
600
Operators are always ___
Repressors are always ____
cis acting
trans acting (for both versions of mutated repressors
600
What happens if glucose is present and lactose are present?
very little lac mRNA
glucose present means no cAMP so activator cap will not bind to the activating sequence, so it cannot help turn on transcription,
But, lactose is there so it gets rid of the repressor from the operator and allows for transcription to happen
So a combination of this will lead to low levels of mRNA
700
What is the lac operon?
The lac operon is a cluster of genes that encode proteins for the uptake and metabolism of lactose, it is an primary example we use to describe prokaryotic gene regulation
700
Lets look at Strain 1: All the letters have a plus, meaning they are all wildtype so those O Z Y genes are doing their job as well and are not mutated. a negative sign means they are mutated. So b-galactosidase enzyme and permease is made as normally as the inducer is present
For strain 2: We notice that there is an F plasmid injected into a bacterial E colu cell that has a mutated Z (because it is negative Z-). the only effect is that the Z-galactosidae protein is mutated from the plasmid but trancription occured as normal.
Strain 3: No plasmid, we just have the DNA of the E. Coli by itself. However, the operator is mutated because it states Oc, so repressor cannot bind and block transcription, so you have transcription occuring when it is not supposed, like when the inducer is not there. That is why there are all pluses.
Strain 4. Plasmid is back and is injected into the E coli. This time we have the F plasmid having a mutated operator, cuasing it to produce b-gala gene even when inducer is not present. However, permease is not express even in the absence of lactose, because it is mutated itself.