nuclear structure and import

Question 1

what are the functions of the nucleus?

Answer 1

• separates DNA from the rest of the cell

- allows for regulation of transcription

Question 2

which processes occur in the nucleus?

Answer 2

• DNA replication

- chromatin organisation and modification

Question 3

what processes does the nucleus regulate?

Answer 3

• DNA replication
• RNA processing
• pre-mRNA splicing
• ribosomal subunit assembly

Question 4

what is the structure of the nucleus?

Answer 4

double membrane continuous with endoplasmic reticulum. contains nucleolus, which is non membrane bound. peripheral heterochromatin is localised at the periphery of the nucleus and is anchored by nuclear lamina

Question 5

what is the role of nuclear pore complexes?

Answer 5

controls movement in and out of the nucleus

Question 6

what is the structure of nuclear pore complexes?

Answer 6

very large, multi-subunit complexes that sit in the double membrane of the nucleus. parts of the complex are inserted into the intermembrane space to anchor the pore in place. the NPC is fairly symmetrical except from cytosolic fibrils which directs proteins through the pore

Question 7

how do NPCs facilitate movement of small proteins into the cell?

Answer 7

the pore is lined with proteins with an extended conformation containing phenylalanine-glycine repeats. these residues aid recognition of proteins which require transport into the nucleus. small proteins can enter the nucleus by diffusion

Question 8

how do NPCs transport large molecules into the nucleus?

Answer 8

active transport
molecules contain sorting signals, recognised by importing and exportins. these transport proteins through the pore against a concentration gradient by GTP hydrolysis

Question 9

what is a sorting signal?

Answer 9

a specific sequence of amino acids that is detected by other cellular components and targets a protein to a specific location

Question 10

what does the classical nuclear localisation signal consist of?

Answer 10

one or two short stretches of basic amino acids

Question 11

how can the function of nuclear localisation signals be measured?

Answer 11

• mutating a residue to a different one of approx same size but lacking positive charge.
• perform immunofluorescence microscopy
• mutants lacking positive charge will not be transported into the nucleus

Question 12

list some examples of proteins which undergo nuclear import?

Answer 12

• structural proteins of the nucleus (histones, nuclear lamins)
• DNA/RNA polymerases
• components of splicing machinery
• ribosomal proteins
• gene regulators

Question 13

what is Ran?

Answer 13

a member of thermal monomeric GTPase family of cellular regulators. molecular switch

Question 14

what is the function of Ran?

Answer 14

allows importins and exportins to sense whether they are in the nucleus or cytoplasm

Question 15

how does Ran act as a molecular switch for importins?

Answer 15

• RanGTP binds to importins, changing their conformation to allow cargo release
• RanGDP dissociates from importins, changing their conformation to allow cargo binding

Question 16

which state is Ran in in the cytosol?

Answer 16

• Ran-GDP predominates (does not bind importin)

- free importin can bind cargo

Question 17

which state is Ran in in the nucleus?

Answer 17

-Ran-GTP predominates

Ran GTP binds importin, a conformational change displaces cargo

Question 18

what is a common nuclear export motif?

Answer 18

leucine rich sequence

Question 19

what are examples of molecules undergoing nuclear export?

Answer 19

mRNA + tRNA

partly assembled ribosomal subunits

Question 20

why is regulation of nuclear transport important?

Answer 20

-regulation of gene transcription
timing of uptake of specific transcriptional regulators
allows quick transcriptional response to external stimuli

Question 21

what type of mechanisms can prevent nuclear import of specific proteins?

Answer 21

masking the NLS via:

• binding of inhibitory proteins
• post-translational modification that causes a conformational change e.g. phosphorylation
• tethering in the cytoplasm

Question 22

give an example of how regulation by phosphorylation can regulate nuclear import?

Answer 22

(SV40 T-antigen) phosphorylation increases the affinity for importin alpha, resulting in increased import into the nucleus