Module 6: Reg. of Gene Expression (2-Component Reg., Chemotaxis) Flashcards
Ttwo-component regulatory systems serve what overall purpose?
To respond to changes in the external environment
Most regulatory systems responding to external environment changes are what?
Two-component regulatory systems
Two-Component Regulatory System
Regulatory system composed of one protein that acts as a sensor and one protein that regulates transcription
What are the two basic components of a 2-component regulatory system?
SENSOR + RESPONSE REGULATOR (RR)
Interaction of sensor and RR results in…
Signal transduction
Signal Transduction
Process producing a cellular response to an external stimulus
What is the most common regulatory system found in bacteria?
Two-component reg. system
Sensor
A protein (typically a KINASE) that DETECTS some environmental stimulus (bind signal molecule)
Kinase
Enzyme that phosphorylates other molecules (or itself) usually through addition of a phosphate group taken from ATP
What is a very commonly used sensor?
Histidine Protein Kinase (HPK)
What is HPK?
Histidine Protein Kinase
== A common sensor kinase that autophosphorylates at a specific histidine residue
–> (Phosphotransfers to specific RR)
Autophosphorylation
Process of protein kinase phosphorylating itself
Response Regulator
RR
== A protein that regulates transcription in a 2 component system
–> Interacts with sensor to activate, usually interaction is a phosphotransfer leading to phosphorylation of the RR
What do RRs interact with?
Interacts with sensor:
–> to activate, usually via a phosphotransfer leading to phosphorylation of the RR
Interacts with DNA and RNA Polym.:
–> To promote/enhance or inhibit transcription of gene/s
General mechanism of 2-component reg. system:
1) External signaling molecule binds to the HPK input domain
2) Binding of HPK triggers autophosphorylation via ATP hydrolysis
== Transmitter domain of HPK is phosphorylated
3) Phosphorylated HPK phosphotransfers to an RR
4) Phosphorylated RR undergoes conformational change
5) Activated RR interacts with DNA and RNA polymerase of a target gene/s
6) Transcription increases or decreases depending on function of the RR
2-component regulatory systems typically do not contain just one sensor and one RR, they usually…
consist of several HPKs and RRs that support an extensive phosphotransfer system
–> Usually a more extensive phosphorylation cascade!
What are 2 examples of a 2 component regulatory system?
1) Bacillus subtilus endospore formation in response to limited nutrient conditions
2) Agrobacterium tumefaciens virulence (of plant tumor genes)
Agrobacterium tumefaciens
A bacterium that causes formation of tumors in plants by transferring tumor formation genes into nuclei of plant cells
What process of A. tumefaciens is a 2 component regulatory system?
The induction and carrying out of of transferring tumor-inducing genes into plant cell nuclei
A. tumefaciens:
What components are needed for the transfer of tumor inducing genes?
1) Ti plasmid (carries vir genes and T-DNA)
2) Inducing conditions: Phenolic compounds and low pH
Ti Plasmid
“Tumor Inducing” plasmid which contains:
1) Tumor formation genes (T-DNA region)
2) Genes needed to mediate transfer of the T-DNA (vir genes)
T-DNA
“transfer DNA”
–> A segment of DNA from the Ti plasmid that gets transferred to plant cells
What genes does the T-DNA of the Ti plasmid contain?
Genes encoding for enzymes that direct plant hormone production
==> Leading to tumor formation!
What are the inducible conditions needed for the 2-component reg. system of A. tumefaciens?
Conditions similar to those present at plant wound site
1) Acidic (low) pH
2) Presence of certain phenolic compounds
What genes does the 2-component reg. system of A. tumefaciens regulate expression of?
vir genes of the Ti plasmid
Inducing Conditions
Stimuli that trigger ACTIVATION of specific genes
The inducing conditions of the 2-component reg. system in A. tumefaciens are technically the inducing conditions of…
THE VIR GENES!
vir Genes
Genes on the Ti plasmid that encode for products that direct the transfer of T-DNA from A. tumefaciens to plant cells
The transfer of T-DNA from A. tumefaciens to plant cells is similar to…
BUT, what is different?
Conjugation!
BUT, only involves the transfer of an excised SEGMENT of plasmid (Ti) NOT the entire thing
In order for T-DNA transfer from A. tumefaciens to plant cells to occur, what must happen first?
Activation of the vir genes!
What genes encode for the components of the 2-component regulatory system in A. tumefaciens?
TWO regulatory genes (outside of vir operon):
1) virA –> Encodes for VirA (sensor, HPK)
2) virG –> Encodes for VirG (RR)
VirA
A transmembrane protein of the HPK family (= SENSOR)
VirG
A transcriptional activator of the RR family
What is the 2 component reg. system process of A. tumefaciens?
1) virA + virG genes constitutively expressed, produce VirA and VirG
2) In presence of inducing conditions, stimulus molecules bind VirA on the input domain
3) VirA autophosphorylates at histidine residue via ATP hydrolysis
4) VirG phosphorylates at aspartic residue via phosphotransfer from VirA-P
5) VirG-P undergoes conformational change, activating it!
6) VirG-P interacts with DNA of Ti plasmid + RNA polymerase
== Activation of vir genes transcription! (activation of vir operon)
== vir gene products formed –> Leading to T-DNA transfer!
How was the sequence of phosphorylation between VirA and VirG determined?
(How did we figure out the order in which phosphorylation occurs)
Through the use of radiolabeled ATP to track phosphorylation
Results of ATP radiolabeling combinations: (3)
VirA + [38P]ATP ==> VirA-38P
VirG + [38P]ATP ==> VirG
VirA-38P + VirG ==> VirG-38P
–> VirA can phosphorylate from ATP directly
–> VirG CANT phosphortlate from ATP directly
–> VirG Can phosphorylate from VirA-P!
Many 2-component regulatory systems involve what phosphorylation path?
Histidine-Aspartate Phosphotransfer!
Histidine-Aspartate Phosphotransfer Pathway
1) ATP is hydrolyzed
2) ATP phosphate is transferred to HISTIDINE residue of HPK sensor
3) Histidine phosphate is transferred to ASPARTATE residue of the RR
HPKs phosphorylate at _______ whereas RRs phosphorylate at ________
HPK phosphorylation == Histidine (His) residue
RR phosphorylation == Aspartate (Asp) residue
What are the domains of an HPK?
2 domains:
N-terminal = Sensor (INPUT) Domain (peaks out into ECF)
C-terminal = Transmitter Domain (peaks out in the cytoplasm)
What are the domains of an RR?
2 domains:
N-terminal = Receiver Domain
C-terminal = ** Output Domain**
On HPKs and RRs where are the phosphorylated His and Asp residues located?
HPK = His residue in Transmitter Domain (C-terminal)
RR = Asp residue in Receiver Domain (N-terminal)
HPK to RR phosphotransfer:
What domains interact?
The transmitter domain of HPK has phosphate on a His residue which phosphotransfers to Asp residue of receiver domain of RR
= Transmitter (C) to Receiver (N)
Phosphotransfer between HPK and RR is determined by…
recognition of HPK transmitter domain and RR receiver domain
(must be cognates)
For interaction to occur between HPK and RR, what must be similar?
The conserved domains (transmitter and receiver domains) must have some level of AA similarity for recognition!
Phosphotransfer between HPK and RR exhibits…
SPECIFICITY
What is meant by:
Specificity of HPK-RR phosphotransfer
Phosphotransfer occurs only between SPECIFIC pairs of HPKs and RRs, NOT other pairs!
In HPKs and RRs, what are the variable and conserved domains?
HPK –>
N-terminal (Input domain) = Variable
C-terminal (Transmitter domain) = Conserved
RR –>
N-terminal (Receiver domain) = Conserved
C-terminal (Output domain) = Variable
Chemotaxis
The movement of motile bacteria TOWARD favorable chemicals (like preferred nutrients) and AWAY from harmful chemicals (toxins, poison, etc.)
The regulation of chemotaxis uses:
A modified 2-component regulatory system that does NOT involve transcriptional or translational control but instead uses protein switches
Regulation of chemotaxis does NOT involve:
Transcriptional or translational regulation
In E.coli:
Clockwise (CW) Rotation =
CW = TUMBLE
–> Peritrichous flagella spread out and individually rotate, causing the cell to spin (resulting in directional change)
In E.coli:
Counter-Clockwise (CCW) Rotation =
CCW = RUN
–> Peritrichous flagella rotate as one bundle causing cell to propel forward in one set direction
In E.coli:
when attractant or repellant is PRESENT, what does this change about rotation and movement?
Changes the ratio of runs and tumbles!
== Changes the ratio of CCW to CW rotation
High attractant conc. OR Low repellant conc. =
CCW rotation is favored!
(> proportion of time spent in CCW rotation)
–> Longer RUN!
Low attractant conc. OR High repellant conc. =
CW rotation is favored!
(> # of CW rotations occurs = > # of tumbles occurs)
–> Shorter runs following high # of TUMBLES
Ultimately, detection of a chemical gradient influences ___________________ which results in chemotactic movement
Detection of a chemical gradient influences the DURATION OF RUNS resulting in chemotactic movement
In chemotaxis, cells move…
UP or DOWN concentration gradients!
Chemotactic Mutants
Cells with disrupted/abnormal chemotaxis
How were chemotactic mutants isolated for study?
By putting cells into a flask containing:
1) Liquid medium, LOW in attractant concentration
2) Capillary tube filled w/ agar, HIGH in attractant concentration
–> WT Cells = respond to the attractant in the tube and move INTO the capillary tube via chemotaxis
–> Mut cells = can’t respond to attractant due to disturbed chemotaxis, causing them to remain in the medium (DONT enter tube)
What phenotype do chemotactic mutants tend to display?
tend to exhibit a “strict” rotational preference:
–> Display a bias towards either CW or CCW rotation! (stuck in a state of longer (CCW) or shorter (CW) runs)
Mutations in what chemotaxis genes cause CCW bias?
What does CCW bias cause?
4 genes:
cheA, cheY, cheW, cheR
CCW bias = Prolonged runs
Mutations in what chemotaxis genes cause CW bias?
What does CW bias cause?
2 genes:
cheB, cheZ
CW bias = More tumbles, shorter runs
Regulation of chemotaxis involves what genes and proteins?
che Genes and resulting Che proteins
==> Make up the modified 2 component reg. system of chemotaxis
Modified 2-component reg. system of chemotaxis involves 2 circuits:
1) Phosphorylation circuit (= responsible for the basis of environmental response; causes changes in rotation)
2) Methylation circuit (= responsible for adaptability, re- and de- sensitization to stimulus)
Che proteins
Products of che genes that are involved in a series of signal transduction pathways that regulate chemotaxis
What are the “central proteins” of chemotaxis?
(What does central protein mean?)
Central protein = che proteins found in ALL chemotactic bacteria
Includes:
1) CheA
2) CheY
CheA
A sensor kinase that autophosphorylates at a histidine residue in the absence or presence of an attractant or repellant
–> One of 3 parts of the sensor complex
CheY
A response receptor (RR)
–> Interacts with CheA to get phosphorylated and then CheY-P interacts with the flagellar motor
Upon phosphorylation of CheY, what happens?
CheY-P goes and interacts with the flagellar motor causing a change to CW rotation = initiates tumble
CheY-P interacts with ___________ NOT ______________
CheY-P interacts with the flagellar motor directly NOT with DNA or RNA polymerase
What is the sensor complex (in chemotactic reg. system)?
(what is it made up of)
The sensor complex is a 3 member complex that is responsible for detecting external stimulus and doing the initial transduction of the signal
Composed of: MCP, CheW, CheA
MCP
Methyl-Accepting Chemotaxis Protein
–> A transmembrane sensory protein that actually binds to attractant/repellant (and transmits the signal to CheW)
CheW
An adapter or coupling protein!
–> Physically connectsMCP to CheA; transduces signals from MCP to CheA
What is the pathway leading the CheA autophosphorylation in the sensor complex?
1) MCP NOT bound by an attractant!
—–> (is either a. unbound altogether or b. bound by repellant)
2) No attractant signal transmitted to CheW = no conformational change
3) CheA autophosphorylates! ==> Triggers cascade leading to a tumble
What is the pathway leading to INHIBITION of CheA autophosphorylation in the sensor complex?
1) MCP is BOUND by an attractant!
2) signal transmitted to CheW = conformational change leading to…
3) CheA is inhibited (no autophosphorylation)
==> Cascade leading to a tumble is inhibited = prolonged Run!
What ultimately determines whether a prolonged run or a tumble initiation occurs?
The state of MCP
–> Whether MCP is bound or not bound by an ATTRACTANT
Relationship between MCP state and run/tumble:
MCP bound by attractant = No CheA-P = Prolonged RUN
MCP not bound by attractant = CheA-P = Tumble initiated
CheZ
A che protein responsible for DEPHOSPHORYLATING CheY-P
== Triggers end of a tumble!
Che reg. system process: Attractant ABSENT (random walk)
1) MCP is not bound by attractant
2) CheW does not conformationally change
3) CheA autophosphorylates! = CheA-P
4) CheA-P phosphotransfers to CheY = CheY-P, conformational change
5) CheY-P interacts with flagellar motor = change to CW rotation
6) TUMBLE
7) (After ~1 sec) CheZ dephosphorylates CheY-P
8) CheY conformationally changes = dissociates from flagellar motor due to loss of phosphate
9) Flagellar motor returns to CCW rotation (run phase)
10) CheY available for phosphorylation again
What is responsible for ENDING the tumble phase?
CheZ!
–> Dephosphorylates CheY-P, triggering it to release from the flagellar motor
Che reg. system process: Attractant BOUND MCP (prolonged run)
1) MCP is bound by attractant
2) CheW gets signal and conformationally changes
3) CheA is inhibited by conformational change = no CheA-P
4) No CheA-P = no phosphotransfer to CheY = no CheY-P
5) No CheY-P = no interaction with flagellar motor
6) Flagellar motor continues its normal CCW rotation = continues RUN
(prolonged due to no signal to change rotation and tumble)
–> CheZ doesn’t do anything because there’s no CheY-P to dephosphorylate
Adaptation (in che pathway)
Another level of chemotaxis regulation that prevents the desensitization of the reg. system to changes in attractant/repellant conc. during prolonged exposure
Adaptation involves what circuit?
The methylation circuit!
Adaptation prevents the regulatory system from getting…
Saturated by repellants/attractants
The methylation circuit of adaptation involves methylation of:
The MCPs!
Methylation of MCP triggers…
Uncoupling of MCP from CheA
== “uncontrolled” CheA autophosphorylation (independent of MCP signal!)
What Che proteins are involved in the adaptation (methylation) circuit?
CheB (demethylates) and CheR (methylates)
CheR
An enzyme that methylates MCP (adds methyl grp)
(triggering uncoupling of MCP and CheA)
Hint: Think methyl grp = R grp = CheR
CheB
A response regulator (RR) that demethylates MCP
–> Gets phosphorylated by CheA-P = CheB-P which undergoes conformational change that activates its ability to demethylate MCP
CheB is a _________ while CheR is NOT, this means that…
CheB is a RESPONSE REGULATOR (RR) while CheR is NOT
–> This means that CheB gets phosphorylated while CheR does not
CheB demethylation of MCP is dependent upon ___________
What is the relationship between CheB demethylation and this factor?
CheA autophosphorylation
Direct (+) relationship:
–> As CheA autophosphorylation increases, CheB demethylation increases
–> As CheA autophosphorylation decreases, CheB demethylation decreases
What is the relationship between MCP methylation and autophosphorylation of CheA?
As MCP methylation increases = CheA autophosphorylation increases (not sensitive to MCP state signal)
As MCP methylation decreases = CheA autophosphorylation decreases (to level proportional to the bound/unbound state of MCP) (sensitive to MCP state signal)
What is the relationship between:
MCP methylation by CheR
Autophosphorylation of CheA
MCP demethylation by CheB
MCP methylation increases = CheA autophosphorylation increases = CheB-P mediated demethylation of MCP increases
MCP methylation decreases = CheA autophosphorylation decreases (to level proportional to MCP state) = CheB-P demethylation of MCP decreases
What is the relationship between:
MCP methylation and demethylation
Why is this important?
Increase in MCP methylation = Increase in MCP demethylation
Decrease in MCP methylation = Decrease in MCP demethylation
–> Important as it ensures that MCP and CheA are not uncoupled for too long, allowing just enough time for desensitization but not too much time that sensitivity is lost for too long
What is the relationship between CheR function and ligand (attractant/repellant) sensitivity?
CheR decreases reg. system sensitivity to ligand (allowing for the system to reset and be able to “check” environment for conc. changes)
–> Uncouples MCP and CheA
What is the relationship between CheB function and ligand (attractant/repellant) sensitivity?
CheB increases reg. system sensitivity to ligand (allowing for the system to “retest” its environment for conc. changes and respond accordingly)
–> Recouples MCP and CheA
What affects extent of CheR methylation of MCP?
The state of MCP (bound or unbound)
–> CheR methylation increases when MCP is bound by a ligand (attractant or repellant)
–> Makes sense because it allows the system to stop responding to that ligand for a second, allowing the ligand time to leave while the system tumbles independently (resetting). Then the system resenstitizes in its unbound MCP state allowing for it to “retest” the environment by seeing if another ligand binds (and then change response accordingly
What is the function of all Che proteins?
CheA = Sensor kinase (HPK)
CheW = coupling protein; transduces signal from MCP to CheA
CheY = Response regulator (RR); Controls flagellar rotation
(phosphorylated state interacts with flagellar motor to trigger CW rotation (tumble))
CheZ = Dephosphorylates CheY-P, ending a tumble cycle
CheR = Methylates ligand-bound MCP
CheB = Response regulator (RR); Demethylates MCP
(in phosphorylated state)
CheA is the HPK to what RRs?
RRs CheY and CheB
–> Phosphorylates these RRs to form active CheY-P and CheB-P
