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SMP - Genetics > Lecture 12 - Signal Transduction-Coupled Factors > Flashcards

Lecture 12 - Signal Transduction-Coupled Factors Flashcards

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Biology 101 flashcards
1
Q

To activate or repress transcription, transcription factors must:

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

Extracellular signals that regulate transcription factor activity must regulate:

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

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

A
  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
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4
Q

Describe the nuclear membrane.

A

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

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5
Q

What are NPCs?

A

Nuclear pore complexes

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6
Q

What is the nuclear lamina?

A

Fibrous meshwork underlying the inner membrane

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7
Q

How many NPCs per cell?

A

3000-4000

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8
Q

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

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

Other parts:

  • Fibrils toward the cytosol
  • Nuclear cage/basket toward the nucleus
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9
Q

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

A

Guide particles toward the pore

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10
Q

How many molecules per second can a nuclear pore transport?

A

500/s

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11
Q

What are the 2 kinds of transport that NPCs facilitate?

A
  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)

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12
Q

Diameter of NPC?

A

50 nm

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13
Q

What mediates nuclear localization?

A

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

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14
Q

What is the role of nuclear import receptors?

A

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

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15
Q

Do we also have nuclear export receptors?

A

YUP

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16
Q

What is Ran?

A

Part of the superfamily of Ras GTPases

17
Q

What is the function of Ran?

A

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

18
Q

How is Ran regulated?

A

Same as Ras

19
Q

Where is Ran found? In what form?

A

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
20
Q

Describe the 5 steps of nuclear import.

A
  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
21
Q

Describe the 4 steps of nuclear export.

A
  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
22
Q

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

A

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

23
Q

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

A
  1. NTF2 binds Ran-GDP in cytoplasm and transports in the nucleus
  2. RanGEF swaps GDP for GTP on Ran
24
Q

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.

A

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

25
Q

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

A

Energy required (ATP) to maintain cellular electrochemical gradients

26
Q

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

A
  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
27
Q

What are FG repeats?

A

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

28
Q

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

A

3D meshwork with hydrogel-like properties

Mutation: selectivity of the pore is lost

29
Q

How are proteins placed IN the nuclear membrane?

A

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

30
Q

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

A

It reveals its NLS

31
Q

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

A
  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
32
Q

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

Example?

A

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

33
Q

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

A

CREB = cAMP response element-binding protein

34
Q

What regulates how genes are regulated by activated transcription factors?

A
  1. Cell-type specific transcriptional regulation
  2. Combinatorial interaction increase specificity
  3. Differential effects of signal strength, such as duration and threshold
35
Q

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?

A

NOPE because there may be other factors expressed interfering with them

36
Q

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

A

YUP and these DNA sequences look very different

37
Q

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

A

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

38
Q

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

A

YUP

39
Q

What is NTF2? Function?

A

Nuclear transport factor 2

Transports Ran-GDP used in nuclear import back inside the nucleus

SMP - Genetics (16 decks)

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