Lecture 12 - Signal Transduction-Coupled Factors

Question 1

To activate or repress transcription, transcription factors must:

Answer 1

1. Be located in the nucleus
2. Bind to DNA and/or
3. Interact with the basal transcriptional apparatus

Question 2

Extracellular signals that regulate transcription factor activity must regulate:

Answer 2

1. Nuclear localization and/or
2. DNA binding and/or
3. Transactivation

Question 3

What are the 7 ways in which signal transduction pathways can regulate transcription factors?

Answer 3

1. Protein synthesis
2. Ligand binding
3. Phosphorylation
4. Addition of a second subunit
5. Unmasking by removing inhibitor
6. Stimulating nuclear entry by removing inhibitor
7. Releasing from membrane

Question 4

Describe the nuclear membrane.

Answer 4

Double-membrane envelope penetrated by pores in which NPCs are positioned and continuous with the endoplasmic reticulum

Question 5

What are NPCs?

Answer 5

Nuclear pore complexes

Question 6

What is the nuclear lamina?

Answer 6

Fibrous meshwork underlying the inner membrane

Question 7

How many NPCs per cell?

Answer 7

3000-4000

Question 8

What are the 4 subunits of a nuclear pore? What 2 other parts are attached to it?

Answer 8

1. Luminal subunit
2. Ring subunit
3. Column subunit
4. Annular subunit

Other parts:

• Fibrils toward the cytosol
• Nuclear cage/basket toward the nucleus

Question 9

What is the purpose of the nuclear fibrils on the nuclear pore?

Answer 9

Guide particles toward the pore

Question 10

How many molecules per second can a nuclear pore transport?

Answer 10

500/s

Question 11

What are the 2 kinds of transport that NPCs facilitate?

Answer 11

1. Free diffusion for small molecules

2. Active transport for macromolecules that are larger than 60,000 daltons (but smaller than 39 nm in diameter)

Question 12

Diameter of NPC?

Answer 12

50 nm

Question 13

What mediates nuclear localization?

Answer 13

Nuclear localization signals (NLS) in the protein sequence: string of basic AAs

Question 14

What is the role of nuclear import receptors?

Answer 14

They recognize and bind the NLS (sometimes via an adaptor) of the cargo protein AND the NPCs

Question 15

Do we also have nuclear export receptors?

Answer 15

YUP

Question 16

What is Ran?

Answer 16

Part of the superfamily of Ras GTPases

Question 17

What is the function of Ran?

Answer 17

Regulation of transport of RNA and protein in and out of the nucleus

Question 18

How is Ran regulated?

Answer 18

Same as Ras

Question 19

Where is Ran found? In what form?

Answer 19

Either side of the nuclear envelop:

• Cytosol: GAP is present so Ran is bound to GDP and inactive
• Nucleus: GEF is present to Ran is bound to GTP and active

Question 20

Describe the 5 steps of nuclear import.

Answer 20

1. Nuclear import receptor binds protein and forms a complex
2. Complex interacts with nuclear fibrils that guide it through the NPC
3. Once in nucleus, Ran-GTPs will compete for the binding site on the nuclear import receptor beta and alpha subunits, thereby releasing the cargo protein
4. Ras-GTPs bound to nuclear import receptor alpha and beta subunits exit the nucleus through the NPC
5. Ras-GTPs encounter GAP which hydrolyzes GTP to GDP + Pi, thereby releasing the alpha and beta subunits

Question 21

Describe the 4 steps of nuclear export.

Answer 21

1. Ran-GTP and protein both bind the nuclear export receptor
2. Complex exits nucleus through nuclear pore
3. Ras-GTP encounters GAP which hydrolyzes GTP to GDP + Pi which also unbinds the protein
4. Nuclear export receptor reenters the nucleus through the pore

Question 22

Describe the structure of the nuclear import receptor. Which part of it binds the cargo protein?

Answer 22

Dimer of importins: alpha (bound to NLS) and beta subunits

Question 23

How is Ran-GDP used in nuclear import brought back in the nucleus?

Answer 23

1. NTF2 binds Ran-GDP in cytoplasm and transports in the nucleus
2. RanGEF swaps GDP for GTP on Ran

Question 24

What site on the beta subunit of nuclear import receptor does Ran-GTP compete for with the cargo protein? Describe the details of how the cargo is released.

Answer 24

Ran-GTP competes with the site bound by the importin beta binding (IBB) domain of the alpha subunit on the beta subunit.
Once IBB portion of alpha subunit unbinds the beta subunit it then competes with the cargo binding domain on the alpha subunit, releasing it

Question 25

What is the energy (GTP) used in nuclear transport comparable to?

Answer 25

Energy required (ATP) to maintain cellular electrochemical gradients

Question 26

What 3 models have been proposed to describe the interior of the nuclear pore? Explain each.

Answer 26

1. Brownian affinity gate model: NPC channel consists of a narrow central tube 2. Selective phase model: the NPC channel represents a selective phase consisting of a meshwork formed by weakly interacting, hydrophobic FG-rich repeats 3. "Oily spaghetti" model: the open NPC channel is filled by hydrophobic, non-interacting FG-repeats that can be pushed aside by receptor-cargo complexes but prevent the passage of other molecules

Question 27

What are FG repeats?

Answer 27

Small hydrophobic segments that break up long stretches of hydrophilic amino acids

Question 28

What do nuclear pore proteins form? What happens if this gets mutated?

Answer 28

3D meshwork with hydrogel-like properties Mutation: selectivity of the pore is lost

Question 29

How are proteins placed IN the nuclear membrane?

Answer 29

Transport from ER membrane through the NPC by usual mechanism (never leave a membrane)

Question 30

How does hormone binding to a particular nuclear receptor affect its translocation to the nucleus?

Answer 30

It reveals its NLS

Question 31

How do the 5 kinases that phosphorylate gene regulatory proteins actually regulate gene expression? 3 ways

Answer 31

1. Phosphorylate cytoplasmic proteins = NLS is revealed 2. Phosphorylate cytoplasmic or nuclear proteins to reveal a DNA binding domain 3. Phosphorylate cytoplasmic or nuclear proteins to active the transactivation domain of the protein

Question 32

What always happens when a protein is translocated to the nucleus? It can be problem. Example?

Answer 32

They will bind to DNA independent of whether that is a productive binding event. Eg: glucocorticoid receptor will bind to DNA in the absence of cortisol

Question 33

What is the name of a protein activated by PKA phosphorylation for gene regulation?

Answer 33

CREB = cAMP response element-binding protein

Question 34

What regulates how genes are regulated by activated transcription factors?

Answer 34

1. Cell-type specific transcriptional regulation 2. Combinatorial interaction increase specificity 3. Differential effects of signal strength, such as duration and threshold

Question 35

If we know that a gene is regulated by a certain set of transcriptional factors, does it mean that this gene will be expressed if they are present?

Answer 35

NOPE because there may be other factors expressed interfering with them

Question 36

Can one transcriptional factor bind different DNA sequences? What to note?

Answer 36

YUP and these DNA sequences look very different

Question 37

What is an example of a DNA binding protein binding 2 different DNA sequences?

Answer 37

Glucocorticoid receptor will either stimulate or inhibit gene expression depending on which DNA sequence it binds

Question 38

Can a specific DNA sequences be bound by more than 1 transcription factor?

Answer 38

YUP

Question 39

What is NTF2? Function?

Answer 39

Nuclear transport factor 2 Transports Ran-GDP used in nuclear import back inside the nucleus