Protein Metabolism - Regulation

Question 1

What is the goal of the Lac Operon/When do you want it to work?

Answer 1

The lac operon wants to metabolize lactose (degrade it) if it is the only preferred fuel source

Question 2

What does the presence of lactose do to the repressor [Lac Operon regulation]

Answer 2

B-gal converts lactose into allolactose. Allolactose inhibits the repressor from binding. Thus, the genes are translated.

Question 3

What does the presence of glucose do to the Lac Operon [Lac Operon Regulation]

Answer 3

Summary: no glucose, yes camp, yes crp, yes translation (vice versa)

Explanation: glucose steals the phosphate groups from a series of enzymes that need to be phosphorylated in

Question 4

What is the goal of the tryptophan operon?

Answer 4

The tryptophan operon wants to make tryptophan when the concentrations of tryptophan drop

Question 5

How does the repressor function depending on tryptophan concentration [TRP operon regulation mechanism]

Answer 5

In the presence of tryptophan, repressor binds, no translation
No tryptophan, no binding of repressor, translation

Question 6

Transcriptional Attenuation [TRP operon regulation mechanism; What happens at low and high tryptophan concentration?]

Answer 6

Transcriptional Attenuation depends on availability of tryptophan charged tRNA and position of the ribosome
Position of ribosome is controlled by two adjacent TRP codons. TRP codons place 2 adjacent tryptophans on a leader peptide.

At high [TRP], ribosome synthesizes leader peptide with no problem. Continues over regions 1 and 2, so 3-4 stem loop forms which is before poly U sequence. Terminates.

At low [TRP], ribosome cannot synthesize leader peptide easily. Ribosome stuck only on region 1, so 2-3 stem loop forms, which does not have a poly U sequence. No termination, Translation continues.

Question 7

4 Iron Homeostasis Proteins

Answer 7

1) Transferrin (import)
2) Ferritin (storage)
3) Aconitase from citric acid cycle (ex. Of usage)
4) Fpn1 (export)

Question 8

Iron Response Element @ 3’-UTR/Which Protein is regulated this way [Consider which protein you want translated depending on the environment]

Answer 8

-Presence of iron, IRP falls off and exposes termination sequence, and gene stops being translated
-no iron, no exposure of termination sequence, gene is translated

Protein: Transferrin. When there’s no iron, you want more iron to be imported

Question 9

Iron Response Element @ 5’-UTR/Which Protein is regulated this way [Consider which protein you want translated depending on the environment]

Answer 9

-Presence of iron, IRP falls off and gene is accessible
-No iron, irp bound and gene is inaccessible

Protein: Ferritin. When there is iron, you can store this iron without starving the cell.

Question 10

R-Protein Operon [Goal of operon? ]

Answer 10

The goal is to stop ribosomal protein synthesis if there is not enough rRNA

Question 11

R-Protein Operon [Which has higher Kd mRNA or rRNA? Which has higher affinity?]

Answer 11

Kd, mRNA > Kd, rRNA. Thus, r-proteins have greater affinity for rRNA.

Question 12

R-Protein Operon [Which Protein is involved?]

Answer 12

Protein L4

Question 13

Translation Riboswitch [How does it work/ When does it work?]

Answer 13

When ligand is present, a secondary structure forms and includes the shine-delgarno sequence, which sequesters it away from the ribosome

Question 14

What happens when elf2 is phosphorylated? What is the name of the kinase and what regulates the kinase [globin protomer synthesis regulation]

Answer 14

When elf2alpha is phosphorylated by kinase HCR, it forms a stable complex of elf2alpha and elf2B. This complex does not translate and does not make globin

heme inhibits kinase and translation is active. Makes sense because if there is heme there needs to be protein.

Question 15

How proteins get into the ER (STEPS DO NOT CORRESPOND TO IMAGE)

Answer 15

1) mRNA is being read by ribosome
2) Signal sequence recognition protein (SRP) binds to the signal sequence . Translation stops because of the bound SRP. (SRP binds stops translation)
3) SRP protein binds to membrane protein on the outer surface of the ER (SRP receptor). This opens a channel called the peptide translocation complex and the peptide chain from the ribosome gets threaded into the ER. (SRP binds receptor and threads protein through channel)
4) SRP protein dissociates because of a GTP timer, and translation can continue (SRP leaves translation continues)
5) Protein is further threaded into the ER
6) Signal sequence is cleaved by a signal peptidase (signal cleaved)
7) Ribosome dissociates from the ER

Question 16

How does Glycosylation happen? [Soluble proteins]

Answer 16

-To glycosylate a protein, carbohydrates are attached to a molecule of dolichol phosphate
-Phosphate of dolichol phosphate points to the cytosol and dolichol points into the ER

1) UDP versions of carbohydrates are activated sugars. Carbohydrates are added to the dolichol phosphate. This step is inhibited by tunicamycin
2) More carbohydrates are attached (adding)
3) Translocation occurs. The dolichol is now facing the cytosol and the phosphates attached to the carbohydrates are facing into the ER. (flipping)
4) More carbohydrates are added
5) Asparagine side chain nitrogen is the nucleophile and the entire glycan is transferred. Dolichol phosphate leaves (protein takes glycan)
6) Molecule of dolichol pyrophosphate is translocated and a phosphate group leaves thus recycling it into dolichol phosphate (dolichol pyrophosphate recycled)

Question 17

How are membrane proteins inserted? What gets stuck in the translocon? [N-terminus inside]

Answer 17

• N-terminus points into the ER lumen and the peptide is threaded through a channel called a translocon
• Movement through the translocon stops when an internal stop sequence (hydrophobic helix) gets stuck within the translocon. This happens because the translocon can only move polar segments all the way through into the lumen.

Question 18

How are membrane proteins inserted? What gets stuck in the translocon? [C-terminus inside]

Answer 18

• This polypeptide chain features an internal signal sequence that cannot be cleaved by a peptidase and remains in the translocon
• Forms a hairpin like shape because the internal signal sequence gets stuck in the translocon
• Ribosome extends the peptide on the C-terminal side and threads it through

Question 19

How do proteins get into the nucleus? [3 steps]

Answer 19

1) two cytosolic proteins (importin-alpha and importin beta) bind to the nuclear localization sequence
2) Complex of the three proteins binds to the nuclear pore complex which catalyzes hte ATP dependent movement into nucleus
3) Importans fall off and protein gets dropped off in the nucleus

Question 20

How are the importins recycled? (how are importins brought back from nucleus to cytosol) 3 steps

Answer 20

1) RAN GTPase binds to the beta importin
2) CAS (cellular apoptosis susceptibility) and RAN GTPase proteins binds to alpha importin
3) Both alpha and beta complex leave the nucleus and with GTP hydrolysis they are dropped off in the cytosol

Question 21

How is the RAN-GDP made functional again?

Answer 21

Meanwhile, Ran-GDP now re-enters the nucleus by binding to nuclear transport factor-2 (NTF2). Once in the nucleus, a specific GEF protein catalyzes the exchange of GTP for GDP, causing NTF2 to be removed from Ran-GTP.

Question 22

How are proteins ubiquinated?

Answer 22

1) ubiqutin gets an adenylyl group added to it (addition of leaving group)
2) E1 displaces leaving group
3) E2 displaces E1
4) E3 displaces E2
5) C-terminus becomes attached to the target protein via a side chain amino group on a lysine present on the target protein

Question 23

High Glucose, Low Lactose [What happens to Lac Operon?]

Answer 23

lac operon off because lac repressor is bound to operator and no C(RA)P protein is bound to to the DNA

Question 24

Low Glucose, Low Lactose [What happens to Lac Operon?]

Answer 24

lac operon off because lac repressor is bound. (if there is no lactose, there is no allolactose)

Question 25

High Glucose, High Lactose [What happens to Lac Operon?]

Answer 25

C(RA)P is not bound so even though lac repressor could dissociate, little lactose enters into the cell and no lac structural genes are transcribed.

Question 26

Low Glucose, High Lactose [What happens to Lac Operon?]

Answer 26

C(RA)P is bound, lac repressor is not bound, lac structural genes are transcribed at a high level.

Question 27

How is avidity achieved by the repressor

Answer 27

There are three operators where the repressor can bind.

Question 28

ER signal properties

Answer 28

• Usually located at the N-terminus
• Typically 12-36 amino acids in length with 10-15 contiguous hydrophobic amino acids

Question 29

Transcriptional Attenuation [TRP operon regulation mechanism; What happens at low and high tryptophan concentration?]

Answer 29

-Transcriptional Attenuation depends on availability of tryptophan charged tRNA and position of the ribosome
-Position of ribosome is controlled by two adjacent TRP codons. TRP codons place 2 adjacent tryptophans on a leader peptide.

At high [TRP], ribosome synthesizes leader peptide with no problem. Continues over regions 1 and 2, so 3-4 stem loop forms which is before poly U sequence. Terminates.

At low [TRP], ribosome cannot synthesize leader peptide easily. Ribosome stuck only on region 1, so 2-3 stem loop forms, which does not have a poly U sequence. No termination, Translation continues.

Question 30

4 Iron Homeostasis Proteins

Answer 30

1) Transferrin (import)
2) Ferritin (storage)
3) Aconitase from citric acid cycle (ex. Of usage)
4) Fpn1 (export)

Question 31

How do proteins get into the nucleus? [3 steps]

Answer 31

1) two cytosolic proteins (importin-alpha and importin beta) bind to the nuclear localization sequence
2) Complex of the three proteins binds to the nuclear pore complex which catalyzes hte ATP dependent movement into nucleus 3) Importins fall off and protein gets dropped off in the nucleus

Question 32

How is the RAN-GDP made functional again?

Answer 32

Meanwhile, Ran-GDP now re-enters the nucleus by binding to nuclear transport factor-2 (NTF2). Once in the nucleus, a specific GEF protein catalyzes the exchange of GTP for GDP, causing NTF2 to be removed from Ran-GTP.