Phys and Genetics Topic 3 Maxine

What major biomolecules in cell contain N?

• Macromolecules

How do cells acquire nitrogen?

1. Organic nitrogen → from enviorment amino acids, nucleic acids, ammonia

2. Inorganic nitrogen → nitrate/nitrite reduced to ammonium, then assimilated into glutamate + glutamine, which donate nitrogen for amino acids, nucleic acids, and other N-containing molecules.

3. Some organisms reduce N₂ gas → ammonium for assimilation.

Available NH3 is taken up via what in Nitrogen assimilation?

• Amt B

What does Nitrogenase do?

• Nitrogen-fixing bacteria and archaea

• converts atmospheric N₂ into NH₄⁺?
  
  

What do Nitrate and Nitrite reductase do?

• convert nitrate/nitrite during nitrogen metabolism

What does Glutamate dehydrogenase, GOGAT, and glutamine synthetase do?

• assimilates NH₄⁺ into glutamate and glutamine?

What does Transaminase/amino transferases do?

• transfer nitrogen from glutamate to other compounds

How is ammonia (NH₄⁺) assimilated using strategy 1?

• 2-oxoglutarate (alpha-ketoglutarate) + NH₄⁺ + NADPH → L-glutamate + NADP⁺ + H₂O, catalyzed by GDH.(Glutamate dehydronase) → L-Glutamate

• NADPH→ NADP + H2O = oxidization

How is ammonia (NH₄⁺) assimilated using strategy 2?

• Step 1: Glutamate + NH₄⁺ + ATP → glutamine via glutamine synthetase (GS).
  Step 2: Glutamine + α-ketoglutarate + NADPH → 2 glutamate via GOGAT.

• Overall reaction : -ketoglutarate + NH4+ + NADPH + ATP + H+
  <==> glutamate + NADP+ + ADP + P

What is the difference between ammonia assimilation Strategy 1 (GDH) and Strategy 2 (GS + GOGAT)?

• Strategy 1 (GDH): Higher Km (~1 mM NH₄⁺) → lower NH₄⁺ affinity; does not require extra ATP.

• Strategy 2 (GS + GOGAT): Lower Km (~1 μM NH₄⁺) → higher NH₄⁺ affinity; can use less abundant NH₄⁺, but costs 1 ATP.= faster one

• Both: Make glutamate from NH₄⁺, NADPH, and α-ketoglutarate.

E. coli is grown in a media containing 2 mM (high conc) of ammonium
chloride. What does this mean?

• Ammonium will be assimilated in the cell through the conversion
  of alpha-ketoglutarate to glutamate

• Ammonium will be assimilated using glutamate dehydrogenase

How are glutamine levels in the cell regulated?

A: Glutamine synthetase (GS) is regulated in 3 ways:

1. Covalent modification: GS is regulated by adenylylation (AMP is covalently attached to a tyrosine residue). at protein level

2. Allosteric regulation: Products of nitrogen assimilation inhibit GS → feedback inhibition. at protein level

3. Transcriptional regulation: glnA gene transcription changes depending on the cellular glutamine concentration. transcriptional level

What is GS (Glutamine Synthetase)?

• GS is a central regulator of N assimilation in many organisms

What is the arrangment/shape of GS?

• its dodecameric w 12 identical subunits arranged as stacked hexagons

How is GS inhibited?

• Allosterically by many end products of N-assimilation (cumulative feedback inhibition)

What does GS produce that also inhibit it?

• GS converts glutamate + NH₃ + ATP → glutamine + ADP + Pi.glutamine makes

• Glycine

• Alanine

• Tryptophan

• Histidine

• CTP

• AMP

• Carbamoyl phosphate

• Glucosamine-6-phosphate

• which eventually inhibit GS

What is allostery?

• Allostery is a process where a molecule binds to one site on a protein or other macromolecule which cahnges its shape or activity of a distanr site

Each active site of GS is near what?

• two Mn2+ ions located at the subunit interfaces

An adenyl transferase (AT) does what to GS?

• adenylates a tyrosine of GS using ATP as AMP donor, releasing pyrophosphate
  (numbering for S. typhimurium). AMP is covalently attached
  to the tyrosine residue

What does Adenylylation of GS do?

Adenylylation of the 12 GS subunits progressively reduces
GS activity (Vmax) up to 30% i.e. enzyme becomes less
active
• The same enzyme can remove adenyl groups from GS by
hydrolysis, releasing adenosine-5-monophosphate (AMP).

What does Adenylylation do to Mn2+?

• changes the divalent cation requirement from Mn2+ to Mg2+.
  • The resulting conformational change increases the sensitivity of GS to cumulative feedback inhibition by its effectors.

What controls the adenylylation state of glutamine synthetase (GS)?

A: The PII regulatory protein controls whether GS is adenylylated or deadenylylated, depending on the cell's nitrogen status.

• High glutamine (N-rich) → PII is deuridylylated → promotes adenylylation of GS → GS activity ↓

• Low glutamine (N-starved) → PII is uridylylated (PII-UMP) → promotes deadenylylation of GS → GS activity ↑

When the [Gln]/[2-Oxoglutarate] ratio is high, is glutamine synthetase (GS) adenylylated?

Yes

When the [Gln]/[2-Oxoglutarate] ratio is low, is glutamine synthetase (GS) adenylylated?

No

What is GS activity like when the [Gln]/[2-Oxoglutarate] ratio is high?

low activity

What is GS activity like when the [Gln]/[2-Oxoglutarate] ratio is low?

High activity

What metal cofactor does GS use when the [Gln]/[2-Oxoglutarate] ratio is high (adenylylated state)?

Mg 2+

What metal cofactor does GS use when the [Gln]/[2-Oxoglutarate] ratio is low (deadenylylated state)?

Mn 2+

Is GS sensitive to feedback inhibition when the [Gln]/[2-Oxoglutarate] ratio is high (meaning glutamine is high)?

Yes

Is GS sensitive to feedback inhibition when the [Gln]/[2-Oxoglutarate] ratio is low (meaning glutamine is low)?

No

Overall, how does adenylylation status relate to GS activity and feedback sensitivity?

The adenylylated form (favored by a high Gln/2-oxoglutarate ratio, using Mg²⁺) has low activity and is sensitive to feedback inhibition; the deadenylylated form (favored by a low ratio, using Mn²⁺) has high activity and is not sensitive to feedback inhibition.

adenylylation of glutamine synthetase leads to what?

• reduced glutamine synthetase activity

• higher sensitivity to feedback inhibition

• will prefer to use magnesium ions as co-factor

Which enzyme senses the level of [Glutamine]/[2-
oxoglutarate] in the cell?

• PII performs this function/ modulates it(GlnB)

What family is PII a part of?

• trimeric proteins

What is the structure of PII?

• 3 identical protomers, each with a flexible “T-loop” which contains exposed tyrosine residues

What is the function of the T-loop in PII?

• Tyrosine in each protomer can be uridylylated to form PII-UMP or deuridylylated
  to form PII.

What is the enzyme that modifies PII?

• uridyltransferase/hydolase

Uridylation and deuridylation of PII are catalyzed by which enzyme?

• a glutamine (Gln)-sensitive uridyl transferase (UTase).

• high [Gln] inhibits its uridyl transferase and activates its uridyl hydrolase activity.
  • i.e. high Gln → PII
  • Low Gln→ PII-UMP

What happens to GS when the [Gln]/[2-oxoglutarate] ratio is high?

• GS is adenylylated (GS-AMP), resulting in low GS activity and increased sensitivity to feedback inhibition.

What happens to PII when the [Gln]/[2-oxoglutarate] ratio is high?

• Glutamine (Gln) binds to uridylyl transferase/hydrolase, increasing its hydrolytic activity.

• PII remains unmodified (PII) and stimulates adenylyltransferase to adenylylate GS→ GS-AMP

What happens to GS when the [Gln]/[2-oxoglutarate] ratio is low?

• GS is deadenylylated (not adenylylated), resulting in high GS activity and no sensitivity to feedback inhibition.

What happens to PII when the [Gln]/[2-oxoglutarate] ratio is low?

• There is less glutamine bound to uridylyl transferase/hydrolase, increasing its uridylyltransferase activity.

• PII is modified to PII-UMP,

• also 2-OG binds to any unmodified PII which prevents activation of adenylyltransferase.

How do different states of PII regulate GS adenylylation?

• Unmodified PII: Stimulates adenylyltransferase, causing GS adenylylation and decreased GS activity.

• PII-UMP: Does not stimulate adenylyltransferase, allowing GS to remain deadenylylated and active.

How does 2-oxoglutarate (2-OG) affect PII?

• 2-OG binds to unmodified PII, preventing it from activating adenylyltransferase, which helps prevent GS adenylylation.

What metal cofactors are associated with GS under high and low [Gln]/[2-OG] ratios?

• High ratio: Mg²⁺

• Low ratio: Mn²⁺

How does feedback inhibition of GS change with its adenylylation state?

• Adenylylated GS: Sensitive to feedback inhibition.

• Deadenylylated GS: Not sensitive to feedback inhibition.

What are the roles of uridylyltransferase (UT) and adenylyltransferase (AT) in GS regulation?

• UT: Adds or removes UMP from PII, depending on nitrogen availability.

• AT: Adenylylates or deadenylylates GS, regulating its activity

What happens to PII when glutamine levels are high?

• High glutamine stimulates the uridylyl hydrolase activity of UT, removing UMP from PII-UMP to form unmodified PII

What happens to PII when glutamine levels are low?

• Low glutamine promotes the uridylyltransferase activity of UT, adding UMP to PII to form PII-UMP.

How does unmodified PII affect adenylyltransferase (AT)?

• Unmodified PII stimulates AT to adenylylate GS, attaching AMP to GS and making it inactive.

How does PII-UMP affect adenylyltransferase (AT)?

• PII-UMP shifts AT activity toward deadenylylation of GS, removing AMP and making GS active.

What happens during GS adenylylation?

• AT uses ATP to attach AMP to GS, releasing pyrophosphate (PPi). This inactivates GS and increases its sensitivity to feedback inhibition.

What happens during GS deadenylylation?

• AT removes AMP from GS using inorganic phosphate (Pi), releasing ADP. This activates GS and decreases its sensitivity to feedback inhibition.

What is the role of α-ketoglutarate (2-OG) in GS regulation?

• α-Ketoglutarate signals nitrogen limitation and binds to PII, helping promote the formation and activity of PII-UMP, which favours GS deadenylylation.

How does high glutamine affect GS activity?

• High glutamine promotes unmodified PII, which stimulates AT-mediated GS adenylylation, resulting in low GS activity

How does low glutamine affect GS activity?

• Low glutamine promotes PII-UMP, which shifts AT toward GS deadenylylation, resulting in high GS activity.

How does PII coordinate nitrogen availability with GS activity?

• High nitrogen availability: Unmodified PII promotes GS adenylylation, decreasing GS activity.

• Low nitrogen availability: PII-UMP promotes GS deadenylylation, increasing GS activity.

What is the overall purpose of regulating GS through PII and AT?

• To match glutamine synthesis to the cell's nitrogen availability by decreasing GS activity when nitrogen is abundant and increasing it when nitrogen is limited.

When [glutamine]/[alpha-ketoglutarate] ratio is high what happens?

(i) PII is not uridylated
(ii) PII stimulate adenylylation of GS
(iii) Adenylylated GS is less active than non adenylylated GS
(iv) Adenylylated GS is sensitive to inhibition by products of
N-assimilation

When [glutamine]/[alpha-ketoglutarate] ratio is low what happens?

(i) PII is uridylated to PII-UMP
(ii) PII-UMP cannot stimulate adenylylation of GS
(iii) unadenylylated GS is highly active

PIIs function vrs PII-UMP

• PII: activate adenyltransferase high glutamine

• PII: does not activate adenyl transferase low glutamine

GS vrs GS-AMP function

• GS-AMP less active more sensitive to feedback high glutamine

• GS: more active less sensitive to feedback low glutamine

What happens if the tyrosyl residue that can be modified by AMP in GS is converted
to phenylalanine by mutating the GS gene ?

GS cannot be modified by AMP -> always active

Can the activity of GS and the effectiveness of PII be
modulated over very broad ranges?

yes because each can be covalently modified to varying degrees

The genetic regulation of glutamine synthetase is controlled by what expression?

• gene A

What is the function of glnK and its protein product PII?

• PII is a nitrogen regulatory protein that senses cellular nitrogen status and regulates nitrogen metabolism through genetic and biochemical effectors.

What is the function of ntrC and its protein product NtrC?

• NtrC is a response regulator that activates the transcription of genes involved in the nitrogen starvation response.

What is the function of ntrB and its protein product NtrB?

• NtrB is a sensor kinase that senses nitrogen availability and regulates NtrC through phosphorylation.

What is the function of rpoN and its protein product σ⁵⁴?

σ⁵⁴ is a nitrogen-related sigma factor that directs RNA polymerase to specific promoters of nitrogen-regulated genes.

What is the function of nifA and its protein product NifA?

• NifA is a response regulator and transcriptional activator that activates the expression of genes involved in nitrogen fixation.

What is the function of nifL and its protein product NifL?

• NifL is a regulatory sensor protein that inhibits NifA under conditions that are unfavorable for nitrogen fixation, such as high fixed nitrogen or oxygen

What is the difference between NtrC and NifA?

• NtrC: Regulates genes involved in the general nitrogen starvation response.

• NifA: Activates genes specifically involved in nitrogen fixation.

What is the difference between NtrB and NifL?

• NtrB: A sensor kinase involved in the NtrB/NtrC two-component regulatory system.

• NifL: A regulatory sensor protein that inhibits NifA to control nitrogen fixation

What are the two promoters for the gln A gene?

• sigma 70 Ap1, Lp :weak low level of expression

• sigma 54: Ap2 strong

What is the function of the Ap1 promoter?

• Ap1 is a σ⁷⁰-dependent promoter that provides basal expression of glnA, allowing some GS to be produced under different nitrogen condition

What is the function of the Ap2 promoter?

• Ap2 is a σ⁵⁴-dependent promoter that is activated by phosphorylated NtrC (NtrC-P), increasing glnA expression during nitrogen limitation.

What is the function of the Lp promoter?

• Lp is a σ⁷⁰-dependent promoter that drives expression of ntrB and ntrC, producing the proteins involved in nitrogen regulation.

What happens to glnA expression when glutamine is abundant?

• Unmodified PII promotes NtrC dephosphorylation through NtrB, reducing activation of the σ⁵⁴-dependent Ap2 promoter. Basal expression from Ap1 continues.

What happens to glnA expression when glutamine is low?

• PII becomes uridylylated, allowing NtrB to promote NtrC phosphorylation. NtrC-P activates the σ⁵⁴-dependent Ap2 promoter, increasing glnA transcription.

Which promoter for glnA is activated during nitrogen limitation?

• The σ⁵⁴-dependent Ap2 promoter, activated by phosphorylated NtrC, leading to increased GS production.

What is read-through of the Rho-independent transcription terminator between glnA and ntrB? How does it happen?

• A: RNA polymerase bypasses the terminator after glnA and continues transcription into ntrB and ntrC, allowing the downstream genes to be expressed from the upstream glnA promoter

• Happens when NtrC-P (gets phosphorylated) and activates Ap2

What is the role of σ⁵⁴-containing RNA polymerase at the glnAp2 promoter?

• RNA polymerase holoenzyme containing σ⁵⁴ can bind to glnAp2, but it cannot form the open complex on its own to initiate transcription.

What is the role of NtrC-P in σ⁵⁴-dependent transcription?

• Multiple phosphorylated NtrC (NtrC-P) molecules bind to upstream enhancer sequences, approximately 100 bp from the promoter, and help activate transcription by interacting with σ⁵⁴-containing RNA polymerase

• needs to be P bc Phosphorylation activates NtrC, enabling it to bind upstream enhancer sequences and stimulate σ⁵⁴-dependent transcription

What is the role of integration host factor (IHF) in σ⁵⁴-dependent transcription?

• IHF bends the DNA, bringing enhancer-bound NtrC-P into contact with RNA polymerase at the promoter to facilitate transcription activation

How does NtrC-P activate σ⁵⁴-dependent transcription?

• NtrC-P binds upstream enhancers and, aided by IHF, interacts with σ⁵⁴-containing RNA polymerase. NtrC-P hydrolyzes ATP to provide the energy needed for open complex formation, allowing transcription to begin.

Transcription of glnA from the sigma 54 promoter is regulated by what 2 component system?

• NtrB and NtrC

• NtrB sensor kinase

• NtrC response regulator

• NtrB recives signal about nitrogen being limited it then autophosphorylates a histidine residue (His 139) using ATP as a phospgate donor, then transfers the phosphate group to an aspartate residue on NtrC, forming NtrC-P.

• NtrC-P then induces expression of sigma 54 promoters

What is the role of NtrB in the two-component system?

• NtrB is a sensor kinase that autophosphorylates and transfers a phosphate group to NtrC in response to nitrogen availability.

What is the role of NtrC in the two-component system?

• NtrC is a response regulator that becomes phosphorylated by NtrB and activates σ⁵⁴-dependent transcription from the glnAp2 promoter.

What is the physiological half-life of NtrC-P?

• NtrC-P is quite labile, with a physiological half-life of 5–10 minutes.

What additional activity does NtrB have?

• NtrB also has NtrC-P phosphatase activity, allowing it to dephosphorylate NtrC-P.

What determines the phosphorylation state of NtrC?

• The balance between the phosphotransfer activity and phosphatase activity of NtrB determines the fraction of NtrC molecules that are phosphorylated

What happens to PII during ammonia excess (high [Gln]/[α-KG])?

• PII is not uridylylated because the deuridylylating activity of UTase is favoured.

How does PII affect NtrB during ammonia excess?(high [Gln]/[α-KG])

• PII associates with NtrB and enhances its phosphatase activity, promoting dephosphorylation of NtrC-P

What establishes basal levels of GS, NtrB, and NtrC?(high [Gln]/[α-KG])

• Transcription from the two σ⁷⁰-dependent promoters, glnAp1 and glnLp, establishes basal levels of GS, NtrB, and NtrC.

How is transcription from glnAp1 regulated by unphosphorylated NtrC?(high [Gln]/[α-KG])

• Binding of unphosphorylated NtrC reduces transcription from glnAp1 by 5-fold.

• glutamine synthesase transcription at sigma 70 is slightly repressed by NtrC

What happens to PII during ammonia limitation (low [Gln]/[α-KG])?

• PII is uridylylated by UTase (GlnD), causing it to release NtrB, which then phosphorylates NtrC.

How is transcription from glnAp2 activated during ammonia limitation?

• NtrC-P oligomers bind to upstream enhancers and interact with promoter-bound RNA polymerase (σ⁵⁴), activating open complex formation and transcription from glnAp2.

What happens when transcription from glnAp2 is activated?

• Transcription from glnAp2 expresses all three genes (glnA, ntrB, and ntrC), causing GS levels to rise 30-fold.

How do NtrC levels change during ammonia limitation?

• NtrC levels rise approximately 24-fold, while transcription from glnAp1 and glnLp is fully repressed.

What happens as glutamine levels rise during ammonia limitation?

• GlnD deuridylylates PII, reversing the process and restoring the nitrogen regulation system toward the ammonia-excess state.