Lecture 3: two component systems Flashcards
Why do bacteria sense their environment?
So that they can respond to certain changes and with that prevent that e.g. their energy supply is used up while in a nutrient scarce environment.
What kind of molecules are sensed by bacteria?
Bacteria sense the availability of carbon/free energy sources like glucose and lactose or the availability of electron acceptors like oxygen and nitrate.
What essential nutrients do bacteria sense and which two of these nutrients are important in the respiratory chain?
They sense ammonium, phosphate, sulphur, iron and copper.
Iron and copper are most important in the respiratory chain.
Why are iron and copper important for the respiratory chain?
They form the catalytic centers of respiratory enzymes.
What else (except nutrients/molecules) can bacteria sense?
The quorum of neighboring cells (single cells, intraspecies, interspecies, host cells).
So to summerize: sum up three things bacteria can sense.
- Sensing the availability of carbon or free energy sources or the availability of electron acceptors.
- Sensing essential nutrients
- Sensing quorum of neighboring cells.
By what two things can metabolic regulation be achieved? Also describe whether this is on protein or DNA level.
- By controlling the activity of an enzyme (protein level)
- By controlling the amount of active enzymes (DNA level)
Just look at this picture and see if you can describe it.
Ok
RNA polymerase (RNAp) is important for transcribing DNA sequences to (m)RNA sequences. What is needed for RNAp to start transcription?
RNAp has a subunit called Sigma factor that binds to specific sequences on the DNA. It ensures that RNAp stably can bind to the DNA.
What sequences are recognized by sigma factor?
Target sequences on -35 (pribnow box) en -10 (TATA box).
Why is it called -35 and -10 sequences and what sequences are these (in E.coli)?
- 10 is -10 nucleotides away from the first mRNA nucleotide that is made.
- 35 –> TTGACA
- 10 –> TATAAT
Sometimes there is no -35 sequence which results in poor binding and release of RNAp. What solution is there?
Binding of RNAp to the DNA can be enhanced by DNA binding activator proteins (protein-protein interaction). An activator is able to bind when it’s activated by an inducer that originates from the environment.
What’s typical about binding of regulatory proteins to the DNA?
That it’s mostly in dimer configuration. One of the dimers will bind to the negative strand and one to the positive strand. This also means that regulatory proteins like activators have a dimer binding site and a DNA binding site.
What is meant by regulation of transcription by positive control?
An activator protein is activated by an inducer (lactose or glucose) so that RNAp can bindt to its promotor on the DNA.
What’s characteristic for the regulatory proteins of transcription by positive control?
That they’re located upstreams (towards the 5’ end)
What is meant by regulation of transcription by negative control?
RNAp is able to bind to its promotor, but downstream (towards the 3’ end) a repressor with a co-repressor is located. These repressors prevent RNAp from starting transcription.
What’s another word for regulation of transcription by negative control?
Steric hindrance
Signal sensing and control of transcription is controlled by regulators. These regulators are either termed one component or two component regulators. What are one component regulators?
These are regulators that are located in the cytoplasm and sense signal molecules either inside the cell (cellular) or molecules that have come from outside the cell (diffused environmental). When one component regulators sense these molecules, it results in a conformational change of the regulator that causes the regulator to bind to the DNA. This causes e.g. transcription of certain genes.
What are examples of one component regulatory systems?
Oxygen sensing by FNR or quorum sensing.
Describe the structure of FNR-type oxygen sensing regulator.
The regulators exists of a few domains (RNAp binding site, a-helical dimerization domain, helix-turn-helix DNa binding site). But most importantly is the 4FE-4S cluster, a little ‘box’ that is oxygen sensitive. This box consists of 4 iron and 4 sulphur and also cysteine residues that make up the structure of the box.
The FNR-type oxygen sensing regulator is only active under anaerobic conditions. Why?
The box or cluster can only be formed when the concentration oxygen is very low. An increase in oxygen will cause the oxygen molecules to bump into the box, which causes the structure to break.
Describe what happens under aerobic and anaerobic conditions in regard to the FNR-type oxygen sensing regulator.
- Aerobic: the regulator is not active, therefore the regulator is a monomer.
- Anaerobic: the regulator is active and can form a dimer with another FNR-type regulator. This makes it possible for the dimer to bind to the DNA, where it acts as an activator for transcription of RNA (also acts as repressor).
Is there a consensus sequence for the binding of FNR dimers?
Yes, it’s TTGaTnnnnATCAA
Under anaerobic conditions: what target genes are there for FNR?
FNR is able to activate and repress certain genes:
- it represses oxidases for aerobic respiration by binding downstreams on the DNA
- it activates/induces reductases for anaerobic respiration by binding upstreams on the DNA.
Just look at the structure of acul homoserine lactones of different bacteria.
Ok
What is diauxic growth?
Cell growth characterized by cellular growth in two phases, and can be illustrated with a diauxic growth curve.
Explain how one component regulation of the lac operon works if glucose has been broken down while lactose is still present.
The CAP (also named CRP) protein (activator) is important in initiation of Lac operon transcription. But can only be active if cAMP is present. cAMP is a product of the enzyme adenylate cyclase, which is only active when glucose and ATP concentration is low. So in absence of glucose, cAMP is present and binds to CAP. This induces a conformational change in CAP which causes CAP to be able to bind to the DNA. This is not enough for transcription of the Lac operon, since LacI represses the operon. Because lactose is still present, it can bind to LacI and causes LacI to fall of the operon. Now transcription and expression of the Lac operon is possible, so that lactose can be broken down.
Signal sensing and control of transcription is controlled by regulators. These regulators are either termed one component or two component regulators. What are two component regulators?
This regulation consists of two regulators: one signal senser (SS) (or histidine kinase (HK)) and one response regulator (RR). The signal senser can pick up on environmental molecules that are unable to pass the cell membrane and hydrophobic molecules in the membrane, this first causes autophosphorylation of SS itself and after also autophosphorylation of RR. RR is activated and can induce gene regulation. Gene regulation can be e.g. the production of a transporter that can transport the molecules that were first unable to pass the membrane into the cell.
What is characteristic for the signal sensor of two component regulation?
The transmembrane signal sensor is a dimer. If two ligands bind to the dimer, it will cause hydrolysis of ATP and phosphorylation of amino acids.
Understand this picture.
Ok
That quinol is sensed by the ArcAB two component regulation system can be made clearest by explaining how quinol is sensed in the absence of oxygen. Explain what happens in the absence of oxygen (anaerobiosis).
No oxygen means that oxidation occurs less compared to reduction of quinol. So therefore the concentration of reduced quinol is much higher compared to the oxidized quinone concentration.
Furthermore, since there’s no oxygen that can be used as electron acceptors for the respiratory chain, ubiquinone is used as an alternative electron acceptor. Ubiquinone has a lower electron output than oxygen and so the concentration quinol remains higher. This results in the reduction of cysteine residues of the signal sensor ArcB and with this activation of signal sensor kinase ArcB. ArcB then phosphorylates the response regulator ArcA. ArcA can subsequently activate anaerobic metabolic genes and suppress aerobic metabolic genes.
If oxygen levels increase, it will also affect the ArcAB system and the ratio quinol:quinone. Explain what will change.
Oxygen levels increase, which will result in a more balanced state of quinol : quinone. This also means that cysteine residues will be partially reduced and partially oxidized (also balanced). This results in the formation of a sulfide bridge between cysteine residues. With this ArcB will also be partially activated which also affects gene regulation.
That quinol is sensed by the ArcAB two component regulation system can also be explained by the mechanism where quinol is sensed in the presence of oxygen. Explain what happens in the presence of oxygen (aerobiosis).
This is almost the opposite of what happens under anaerobic conditions. Since oxygen is now fully present, oxidation can also happen fully. This means that there’s a high concentration of oxidized ubiquinone compared to reduced ubiquinol. This results in oxidation of all cysteine residues, which results in formation of a disulfide bridge. Here, the signal sensor ArcB is inactive. Gene regulation will now be active in regard to aerobiosis and inactive in regard to anaerobiosis.
Phosphate molecules cannot pass the membrane, but are of course needed in the cell. So phosphate also makes use of a two component system (PhoR/PhoB system). Explain what happens when the concentration of phosphate is high.
While phosphate cannot pass the membrane in general, it’s able to pass a fraction of phosphate molecules A high concentration of phosphate results in a fraction of phosphate molecules still being able to pass the membrane. Here phosphate molecules bind with SS PhoR which keeps the SS inactive. The PhoR/PhoB system is then inactive.
Phosphate molecules cannot pass the membrane, but are of course needed in the cell. So phosphate also makes use of a two component system (PhoR/PhoB system). Explain what happens when the concentration of phosphate is low.
When phosphate concentration is low, few to no phosphate molecules will pass the membrane. This means that SS PhoR lacks binding of a phosphate molecule, which results in autophosphorylation of PhoR and subsequently also PhoB. PhoB is now active, which is then able to bind to target operons.
What genes get upregulated when the concentration of phosphate is low and the PhoR/PhoB system is active?
Genes that are important for the uptake of phosphate or increase of phosphate concentrations. E.g. alkaline phosphatases (enzyme that splits phosphate groupes from molecules), porins (important for phosphate uptake) or a GA-P transporter (the phosphate group can be taken off).
Look at this picture and try to find:
- the basal body or motor
- the rotational part
- the propeller
- the basal body is embedded in the cell wall, beggining within the cytoplasm and ending at the outer membrane.
- the rotational part is located in the outer membrane
- the propeller is located outside the outer membrane
What is the difference between when phosphorylated CheY translocates and binds to the flagellar motor and when CheY isn’t phosphorylated and not translocated to the flagellar motor?
When CheY-P has translocated to the flagellar motor it initates clockwise rotation of the tail of the flagella. It also means that there’s no attractant present. When CheY is present, not phosphorylated and not translocated to the motor, the tail of the flagella rotates counterclockwise. This means that an attractant is present.
How is signal termination achieved when CheY is active and bound to the flagellar motor and there’s tumbling?
CheZ (phosphatase) can dephosphorylate CheY and CheA can also slowly phosphorylate and activate CheB. CheB is a demethylase that takes off the methyl group that is activating the receptor and CheA.
What happens when a chemoattractant binds to the transmembrane chemoreceptor?
Binding of an attractant induces a conformational change in the chemoreceptor. This represses autophosphorylation of CheA and results in CheB dephosphorylating CheA. Therefore ChY-P is released from the flagellar motor and counterclockwise rotation and running to the ligand is initiated.
