The Nucleus Flashcards
What are the 7 main structures of the nucleus?
nuclear envelope
lamina
nuclear pores
matrix
nucleoplasm
perinuclear space
chromatin
T or F: all DNA is located in the nucleus
FALSE, some DNA is in the mitochondria and chloroplasts
About what percent of a cell’s volume does the nucleus occupy?
~10%
Describe the nuclear envelope
2 membrane bilayers separated by a 10-50 nm space (perinuclear space) that surrounds the nucleus
Each membrane has its own complement of proteins
Describe the perinuclear space
the 10-50 nm space between the two membrane bilayers of the nuclear envelope
How many membranes does the nuclear envelop consist of ?
2 membrane bilayers
What structure is the perinuclear space continuous with?
the ER lumen
The outer nuclear membrane is continuous with which structure?
the ER membrane
T or F: the rough ER membrane is the only membrane that can be have ribosomes attached
FALSE, the outer nuclear membrane can also be studded with ribosomes
Where are the two membrane bilayers (nuclear envelope) fused?
at nuclear pores
Describe nuclear pores
protein complexes that are the entry and exit of the nucleus
How many nuclear pores does the average mammalian cell contain?
thousands
Describe the nuclear lamina
A thin, filamentous meshwork on the inner surface of the nuclear envelope in animal cells
What is the function of the nuclear lamina?
Structural support for the nucleus, especially the envelope
to keep it round
What are the filaments of the nuclear lamina made of?
Lamina proteins
How is the lamina network assembled and disassembled?
by lamin phosphorylation
Why would the lamina network need to assemble and disassemble?
lamina would need to disassemble for cell division to separate cells and then would need to be reassembled in each new cell
During interphase, what 4 things would we see in the nucleus?
- nuclear matrix
- nucleoplasm
- chromosomes
- nucleolus
What is the nucleoplasm?
the fluid inside the nuclear envelope
What would the structure of chromosomes be during interphase?
highly extended fibres with both DNA and chromatin protein
Describe the nucleolus
an irregularly shaped electron dense region in the nucleus that functions in ribosomal RNA and ribosome synthesis
What is the function of the nucleolus?
ribosomal RNA and ribosome synthesis
Describe the nuclear matrix
a dynamic meshwork of insoluble filamentous scaffolding proteins within the nucleus
What is the nuclear matrix analogous to?
cytoskeleton in the cytosol
What is the function of the nuclear matrix?
to compartmentalize regions of the nucleus by binding to chromatin
What does assembly and disassembly of the nuclear matrix depend on?
phosphorylation
What 7 things does the nucleoplasm contain?
ions
nucleotides
phosphates (and other components of DNA and RNA)
RNA molecules (ex. RNA transcripts, rRNA, RNPs)
enzymes and proteins
CHROMATIN
1 micrometer of mitotic chromosome length typically contains how many cm of DNA? How much condensation is this?
1 cm of DNA
this is a 10,000 fold condensation
How many meters of DNA does the average cell contain?
2 meters!
T or F: chromosomes are more condensed in interphase than during mitosis
FALSE FALSE FALSE
way more condensed during mitosis
Describe eukaryotic chromatin
Composed of DNA, histone proteins, and non-histone chromosomal proteins in almost equal parts
What does prokaryotic DNA lack that eukaryotic chromatin has?
lacks histone proteins
Describe histones
proteins in eukaryotic chromatin that condense DNA so it will fit into the nucleus
How many major classes of histones are there? What are they?
5
H1 H2A H2B H3 H4
Describe the structure of histones
relatively small proteins with a lot of basic (POSITIVELY CHARGED) residue amino acids
Are histones positively or negatively charged?
positively charged
How does the charge of histones help them coil DNA?
DNA is negatively charged due to its sugar-phosphate backbone and histones are positively charged due to their basic residues so attractive forces help histones coil DNA
What is the basic packing unit of chromatin?
a nucleosome
Describe a nucleosome
DNA that is coiled around a histone protein core
How is a nucleosome formed?
DNA loops around a histone core twice (~150 base pairs of DNA coil around the histone)
~50 nucleotides act as a spacer between histones
Histone 1 will bind to the linker DNA
How many nucleotides and histones will 1 nucleosome involve?
200 nucleotides/base pairs
8 histones
What is the first level of coiling DNA? Describe it
nucleosomes form ‘beads on a string’
Histones are the beads and the DNA is the string
What is a key feature of histones?
they have long nitrogen-terminal tails that project outwards to interact with one another and aid in condensing even more
What is the second level of condensing?
solenoid
Describe the solenoid shape
Nucleosomes are coiled further into the solenoid shape which represents a wound garden hose
aka DNA already coiled around histones is coiled again into the solenoid
How thick is the fibre in the solenoid shape?
30 nm
What is the third level of condensing?
Loops
How is the third level of condensing achieved?
Chromatin fibres wrapped in the solenoid are coiled with NON-histone proteins (scaffold proteins) into loops and anchored onto the scaffold protein
What kind of protein is the solenoid structure coiled with and anchored to?
NON-histone scaffolding proteins
What is the fourth structure coiled chromatin makes?
the condensed mitotic chromosome
What are the 2 types of chromatin in the interphase nucleus?
euchromatin
heterochromatin
Describe euchromatin
loose chromatin ready for gene transcription
appears lighter on a micrograph
Describe heterochromatin
densely packed chromatin that cannot be transcribed
appears darker on a micrograph
What are the two kinds of heterochromatin?
constitutive
facultative
Describe constitutive heterochromatin. Give examples
heterochromatin that is always condensed in all cells
it is permanently silent
ex. the centromere and telomeres
Describe facultative heterochromatin
heterochromatin that is specifically inactivated in certain life stages or in certain cells
Describe epigenetic inheritance
the inheritance of phenotypic changes that do not result from changes in the nucleotide sequence of DNA
How does an epigenetic change influence phenotypic changes?
it alters how DNA code is read
what regulates epigenetic changes?
environmental factors, behaviour, nutrition, etc.
Give a specific example of epigenetic inheritance
people born from mothers who were pregnant during the Dutch Winter Famine of 1944-1945 were more likely to develop heart diseases, schizophrenia, and type 2 diabetes because of the changes in methylation of certain genes
Explain the effects on the people who were born from pregnant mothers during the Dutch Winter Famine of 1944-1945
pregnant mothers during a famine would be receiving less nutrition and therefore so would the baby
these people were more likely to develop heart diseases, schizophrenia, and type 2 diabetes due to epigenetic changes
Describe the histone code
An epigenetic change that causes the disordered N-termini of histones to be phosphorylated, methylated, acetylated, glycosylated, etc.
these modifications are passed on to daughter cells because of replication and division
What does the histone code hypothesis suggest?
the activity of a particular chromatin region depends on the specific modifications to the histone tails (N-termini) in that region
Explain how histone modifications affect the degree of compaction
epigenetic changes to the histone code can cause heterochromatin which is harder to unpack to inactivate DNA that is not likely to be transcribed
What is an example of facultative heterochromatin
calico cats
females have to inactivate one X chromosome randomly (facultative heterochromatin) =
some patches of the skin will inactivate brown colour –> orange patch
some will inactive orange –> brown etc.
T or F: a huge amount of traffic moves into and out of the nucleus through pores
true
How are replication and transcription related to nuclear pores
they require a lot of proteins that are synthesized in the cytoplasm to be transported into the nucleus via the pores
How do mRNA, tRNA, and ribosomal subunits relate to nuclear pores?
they are manufactured in the nucleus and need to be transported out into to cytosol via the pores
T or F: some cell components are synthesized in the nucleus, assembled into a functional unit in the cytoplasm, and shipped back into the nucleus to function
true
How many macromolecules per second can each nuclear pore transport?
up to 1000 macromolecules a second
T or F: each pore transports macromolecules in only one direction at a time
FALSE. pores move molecules in both directions at the same time
What kind of technique would be good to visualize nuclear pores?
gold particles with an electron microscope
the particles can be seen moving into the nucleus through the pores
What do nuclear pores contain that project into the nucleus and the cytoplasm?
a nuclear pore complex
How many proteins make up a nuclear pore complex? are they all the same?
~30 different proteins
What are the proteins called that make up a nuclear pore complex?
~30 nucleoporins make up the NPC
What kind of symmetry does the overall nuclear pore complex have?
octagonal symmetry
What kind of symmetry does each nucleoporin have?
octagonal symmetry
How does the size of a nuclear pore complex compare to a ribosome?
~30x the size of a ribosome
Describe the structure of a nuclear pore complex
a complex of about 30 different proteins (nucleoporins) with octagonal symmetry (each nucleoporin is also has octagonal symmetry) and it has a large, expandable central channel (the pore)
What kind of molecules can move through the central channel/pore of a nuclear pore complex passively?
small polar molecules
T or F: the central channel/pore of a nuclear pore complex can move fully folded proteins - why/why not?
true because it is large and expandable
What kind of structure do some nucleoporins contain?
FG (phenylalanine/glycine) repeats of disordered structure along the lining of the channel
What purpose do the FG domains of disordered structure serve?
they line the channel/pore to form a hydrophobic sieve to block the passive diffusion of large molecules through the pore/channel
Does the FG domain create a hydrophilic or hydrophobic region in the nuclear pore? why does it do this?
hydrophobic
to block the passive diffusion of large molecules
regulates large protein movement
What is FG?
repeats of 2 amino acids:
phenylalanine (F)
glycine (G)
What must proteins destined for the nucleus possess in order to be moved into the nucleus?
a nuclear localization signal (NLS)
What is a nuclear localization signal (NLS)?
A small, specific amino acid sequence (~6-7) (within a larger protein) that targets a protein to move into the nucleus
describe an NLS
a small series (6-7) of positively charged amino acids within a larger protein that will target a protein to move to the nucleus
acts as a signal to move a protein into the nucleus
Describe an importin
A family of proteins that binds to a nuclear localization signal on another protein
What will an NLS on a protein bind to in the cytoplasm?
Importins
What can each importin bind to?
A subset of ‘cargo’ proteins destined to the nucleus
What is a cargo?
A protein that has an NLS and will bind to an importin to move to the nucleus
Where are importins located?
in the cytoplasm
What do importins carrying cargo interact with in the cytoplasm?
the fibrils extending into the cytosol from the nuclear pore complex
How do importins move from the cytosol into the nucleus?
When they are carrying cargo, the importins will form temporary bonds with one FG domain (of the nuclear pore complex channel) and then another and another and move into the nucleus
What happens once the importin and its cargo are inside the nucleus?
the importin dissociates from the cargo
Describe the structure of an importin
Alpha helix and beta sheet subunits of importin form a dimer (CHECK THIS)
What kind of structure does an importin have?
A dimer (2 subunits) 1 alpha helix + 1 beta sheet (is it a beta sheet??)
Once the dimer is formed, what can the importin then do?
Bind to the cargo protein
What happens after an importin binds to a cargo protein?
Importin ‘docks’
Binds to the cytoplasmic filaments of the nuclear pore complex
What kind of micrograph can be used to see importin with cargo binding to the cytoplasmic filaments?
gold particles and an EM
What happens after importin binds to the cytoplasmic filaments of the nuclear pore complex?
The filament that importin has bonded to undergoes a conformational change and folds inwards into the pore
What is the purpose of importin binding to the cytoplasmic filaments of the NPC?
when it binds to the filaments, a conformational change occurs and the filaments fold into the pore, bringing the importin + cargo into contact with the FG domains
T or F: after the importin dissociates from the cargo protein in the nucleus, the importin dimer stays in the nucleus
False
the importin dissociates from the cargo protein and must return to the cytosol to bring more cargo in
Describe Ran GTPase
a monomeric protein that exists in 2 conformations that depends on whether GDP is bound or GTP is bound
What subunit of importin will Ran bind to?
the beta importin
What are the 2 conformations of RanGTPase? What do these depend on?
On = bound to GTP Off = bound to GDP
What does Ran binding to the beta importin do?
Separates all the components of the importin
cargo
beta importin
alpha importin
all separate
How many conformations does Ran GTPase have?
2
T or F: alpha and beta importin move back to the cytosol together - why/why not?
False
When Ran binds to the beta importin, the components all separate so beta and alpha importin move back to the cytosol separately
Why does Ran GTPase act like a molecular switch?
they have an active ‘on’ conformation and an inactive ‘off’ conformation
What is GTP?
Guanine triphosphate
What is GDP?
the hydrolyzed version of GTP
Guanine diphosphate
What kind of structure does an Ran GTPase have (monomer, dimer, trimer…)?
Monomer
T or F: the 2 conformations of Ran GTPase are found in the same location in the cell
False
Where is the GTP-bound Ran GTPase more commonly located in the cell?
in the nucleus
Where is the GDP-bound Ran GTPase more commonly located in the cell?
in the cytosol
How does Ran GTPase switch from ON to OFF (ie., between conformations)?
GAP activates the ability of the Ran GTPase to hydrolyze GTP to form GDP and turn itself off
Describe GAP and its function
GTPase Activating Protein that activates the ability of Ran GTPase to hydrolyze GTP to form GDP and turn off
What does GAP stand for?
GTPase Activating Protein
How does Ran GTPase switch from OFF to ON (ie., between conformations)?
GEF exchanges GDP for another new GTP
Describe GEF and its function and what it stands for
Guanine nucleotide Exchange Factor
A protein that exchanges GDP for a new GTP
Explain why Ran GTP is more common in the nucleus
When Ran GDP enters the nucleus it is quickly converted to Ran GTP by GEF and remains that way until it leaves the nucleus
Explain why Ran GDP is more common in the cytosol
As soon as Ran GTP enters the cytosol, GAP converts it to Ran GDP
In the nucleus, is Ran GTPase on or off? why?
ON
It will be converted from Ran GDP to Ran GTP by GEF
In the cytoplasm, is Ran GTPase on or off? why?
OFF
it will be converted from Ran GTP to Ran GDP by GAP
What does beta importin need to bind to in the nucleus after separating in order to return back to the cytosol?
the Ran GTP in the nucleus
Describe how beta importin moves back to the cytosol from the nucleus
Beta importin binds to Ran GTP in the nucleus and moves with it through the nuclear pore
Ran GTP will be hydrolyzed in the cytosol to Ran GDP and will release the beta importin
What is the importance of the conformational change from Ran GTP to Ran GDP when it enters the cytosol?
Ran GTP will be carrying beta importin with it through the nuclear pore
In order to release it, Ran GTP must be hydrolyzed to Ran GDP
What is an NES?
Nuclear Export Signal
What is an exportin?
A protein that binds to a protein with an NES signal in the nucleus
Where are exportins mostly located?
in the nucleus
What function does exportin have as part of the Ran cycle?
Exportin binds to Ran GTP and can bind with alpha importin and move the alpha importin through the nuclear pore and into the cytosol
How does alpha importin return to the cytosol?
An exportin protein binds to the Ran GTP in the nucleus and the alpha importin and they all move into the cytosol and will be released when Ran GTP is hydrolyzed
How is the alpha importin released from the exportin when they enter the cytosol?
the exportin is also bound to Ran GTP so when they enter the cytosol, Ran GTP will be hydrolyzed to Ran GDP and the conformational change will cause the alpha importin to be released
Briefly describe the steps of transporting proteins into and out of the nucleus
- An importin (made of alpha and beta) protein binds to a protein with an NLS
- importin + cargo bind to the cytosolic filaments of the NPC causing the filaments to bend in
- importin now able to transiently bind to the FG domains of the nuclear pore and can move into the nucleus with the cargo
- Ran binds to the beta importin causing the alpha, beta and cargo to separate
- beta importin binds to Ran GTP and when they enter the cytosol, Ran GTP is hydrolyzed by GAP into Ran GDP and releases beta importin
- exportin proteins binds to an NES on a random protein, then it binds to Ran (Ran has alpha importin as a substrate)
- when they enter the cytosol, Ran GTP is converted to Ran GDP by GAP and releases the alpha importin
