Lecture 3: Chromatin and Nuclear Export

MCB 104

T or F: Energy for the directed movement of proteins into and out of the nucleus is primarily provided by ATP hydrolysis.

False

T or F: The nuclear and cytoplasmic sides of the nuclear pore complexes are structurally distinct, which is important for the pore function

True

Ran GTPase regulates export by binding ________ proteins.

exportin

List the Exportin Steps

1. Ran-GTP binds NER, forming a complex (in the nucleoplasm).

2. The NES region of cargo binds to NER

3. The NER interacts with the FG-nucleoporins in the NPC core, and the complex passes through the basket (to the cytosol)

4. GTP is hydrolyzed to GDP, and Ran-GDP dissociates from the NER

5. The affinity of the NER for cargo is reduced, leading to the release.

6. Ran-GDP is ready to be recycled

List Key Features of Protein Export

• Exportins - nuclear export receptors

• Exportins are closely related to importins; members of the karyopherin family of proteins

• Has a NER (nuclear export signal)

• Ran facilitates export as well as cargo release in the cytosol

3 Similarities of Import and Export

1. Use the nuclear pore complex (NPC)

2. Use Transport receptors (both need karyopherin-family proteins)

3. Depend on the Ran GTP/GDP gradient

   1. Ran-GTP high in nucleus

   2. Ran-GDP high in cytoplasm

• Ran localization determines direction

4. Cargo release happens because receptor affinity changes in different compartments

3 Differences of Import and Export

1. Ran-GTP causes cargo release in import, but cargo binding in export

2. Complex in Export is 3 proteins, for import is 2 proteins

3. Importin has an NLS signal; Exportin has an NES signal

What happens if Ran-GTP cannot be hydrolyzed?

Import: Cargo can still unload in the nucleus, but receptor recycling gets messed up

Export: Cargo may get stuck on exportin because hydrolysis is required for release into the cytoplasm.

General Levels of Chromatin Organization

1. Level one: 11 nm fiber

2. Level two: 30 nm fiber

3. Looped domains

Describe Level One of Chromatin Organization

• DNA wraps around histone octamer

• wrapped DNA + histone core = nucleosome

• Linker DNA connects neighboring nucleosomes

• Histone tails sticks out for PTMs

Think of beads in a string

Describe Level Two of Chromatin Organization

• Cell compacts the beads-on-string into a thicker fiber

• Transition mediated by H1 histone

Describe Level Three of Chromatin Organization

• 30 nm gets folded into loops, mainly by cohesin complexes in interphase

What is H1’s role

• NOT part of the nucleosome core

• Sits on top of DNA, binds linker DNA, pulls neighboring nucleosomes together, and helps tighten the chromatin fiber

H1 = linker histone = promotes higher-order packing

What is Cohesin’s role?

It is an SMC complex (Structural Maintenance of Chromosomes)

• Forms a ring that can trap DNA, create looped domains

What protein mediates transition from 11 nm to 30 nm?

H1

What protein mediates loop formation in interphase?

Cohesin

How does acetylation affect accessibility?

loosens histone-DNA interaction —> more transcription

Describe 3 ways 11 nm fiber chromatin remodels

1. Nucleosome sliding - same nucleosome, different address

2. Nucleosome eviction - remove nucleosome entirely; more open DNA = more likely active transcription

3. Histone exchange - same position, different histone personality

A transcription factor suddenly gains access to a promoter without histone loss. What likely happened?

nucleosome sliding

mRNA’s are synthesized bu ____

RNA Pol II

When and what does 5’ capping occur?

Very early during active transcription. Protects from degradation, acts as a “self” identity signal

Functions of poly a tail

increases stability, helps export

What protein recognizes the 5’ cap for mRNA export?

CBP (cap binding protein)

What does CBP do during export?

Binds 5’ cap and helps recruit the mRNA export machinery at the nuclear pore

What happens to CBP after export?

It is exchanged for translation initiation factors in the cytoplasm

Why is protein exchange after export important?

It converts the transcription from an export-ready mRNA into a translation-ready mRNA.

Why is mRNA export tightly linked to capping and processing?

Only properly processed “self” mRNAs should leave the nucleus, ensuring incomplete RNAs are not translated.

Is mRNA export Ran-dependent like protein importin/exportin?

No - mRNA export is mainly Ran-INDEPENDENT

Walk the full pathway from transcription to translation-ready mRNA

RNA Pol II transcription → 5’ capping → splicing → polyadenylation → CBP binds cap → nuclear export → CBP exchanged for initiarion factors → translation