nuclear structure and import Flashcards
what are the functions of the nucleus?
- separates DNA from the rest of the cell
- allows for regulation of transcription
which processes occur in the nucleus?
- DNA replication
- chromatin organisation and modification
what processes does the nucleus regulate?
- DNA replication
- RNA processing
- pre-mRNA splicing
- ribosomal subunit assembly
what is the structure of the nucleus?
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
what is the role of nuclear pore complexes?
controls movement in and out of the nucleus
what is the structure of nuclear pore complexes?
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
how do NPCs facilitate movement of small proteins into the cell?
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
how do NPCs transport large molecules into the nucleus?
active transport
molecules contain sorting signals, recognised by importing and exportins. these transport proteins through the pore against a concentration gradient by GTP hydrolysis
what is a sorting signal?
a specific sequence of amino acids that is detected by other cellular components and targets a protein to a specific location
what does the classical nuclear localisation signal consist of?
one or two short stretches of basic amino acids
how can the function of nuclear localisation signals be measured?
- 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
list some examples of proteins which undergo nuclear import?
- structural proteins of the nucleus (histones, nuclear lamins)
- DNA/RNA polymerases
- components of splicing machinery
- ribosomal proteins
- gene regulators
what is Ran?
a member of thermal monomeric GTPase family of cellular regulators. molecular switch
what is the function of Ran?
allows importins and exportins to sense whether they are in the nucleus or cytoplasm
how does Ran act as a molecular switch for importins?
- RanGTP binds to importins, changing their conformation to allow cargo release
- RanGDP dissociates from importins, changing their conformation to allow cargo binding
which state is Ran in in the cytosol?
- Ran-GDP predominates (does not bind importin)
- free importin can bind cargo
which state is Ran in in the nucleus?
-Ran-GTP predominates
Ran GTP binds importin, a conformational change displaces cargo
what is a common nuclear export motif?
leucine rich sequence
what are examples of molecules undergoing nuclear export?
mRNA + tRNA
partly assembled ribosomal subunits
why is regulation of nuclear transport important?
-regulation of gene transcription
timing of uptake of specific transcriptional regulators
allows quick transcriptional response to external stimuli
what type of mechanisms can prevent nuclear import of specific proteins?
masking the NLS via:
- binding of inhibitory proteins
- post-translational modification that causes a conformational change e.g. phosphorylation
- tethering in the cytoplasm
give an example of how regulation by phosphorylation can regulate nuclear import?
(SV40 T-antigen) phosphorylation increases the affinity for importin alpha, resulting in increased import into the nucleus
