Lecture 4: Nucleus Flashcards
Parts of the Nucleus
- Nuclear Envelope
-Nuclear Pore - Nucleoplasm
-Nucleolus - DNA (Chromatin)
-made of phospholipid bilayer
-separates cytoplasm and nucleoplasm
-Inner and outer membrane separated by perinuclear space
-Outer nuclear membrane continuous with RER and studded with ribosomes
Nuclear envelope
a network of protein fibers that support and organize the chromatin and other structures
Nuclear Matrix
2 types of Chromatin
-Active Chromatin
-Inactive Chromatin
Appear Lighter
Active Chromatin
Active Chromatin
euchromatin
is darker
and located near periphery
of nucleus
Inactive chromatin
Inactive chromatin
heterochromatin
*a granular region
*site of ribosome
assembly
Nucleolus
two types of heterochromatin
-Constitutive heterochromatin
-Facultative heterochromatin
- Regions that are always heterochromatic
- Permanent part of the genome and not convertible to euchromatin
- Permanently inactive with regard to transcription
Constitutive heterochromatin
- Regions that can interconvert between euchromatin and
heterochromatin - Takes on staining and compactness characteristics of heterochromatin
during some phases of development - Example: Barr body
Facultative heterochromatin
Heterochromatin lies against the __________ in patches and is broken up at the site of the __________.
-Nuclear Envelope
-Nuclear pore
- Results from packaging of
chromatin - Transcription stops
- Chromosomes visible in
microscopy - Separation of haploid
chromosome sets occurs in
nuclear division - Packaging ratio about 10000:
CHROMATIN CONDENSATION
basic structural unit of eukaryotic
chromosome
Nucleosome
Nucleosome is an octamer composed of two each of __, ___, ___, and ___ histone proteins
H2A, H2B, H3 and H4
DNA wrapped around octamer and H1 stabilizes association of nucleosomes to form _________
solenoid
A nucleosome is a complex of a _______ and _____________
histone and 146 nucleotide pairs
-highly conserved sequences (universality in eukaryotes)
- H3, H4 first to form, tightly bound, most conserved
- H2A, H2B, well conserved with species-specific variation
-H1 divergent, absent in S. cerevesiae
Histones
The compaction of linear DNA in eukaryotic chromosomes involves interactions between ______ and ______________
DNA and various proteins
Proteins bound to DNA are subject to change during the life of the cell. These changes affect the _______________________
degree of chromatin compaction
Proteins are brought into the nucleus from the cytoplasm, and can be sent out too by __________
Nuclear Pore
RNAs (messenger RNA, ribosomal RNA and transfer RNAs) are all transported out of the nucleus but only when they are _________
completed
– a specific amino acid sequence marks protein for nuclear entry (Laskey, 1982)
– a series of positively charged amino acids in specific sequence: (pro – lys – lys – lys – arg – lys – val –)
Nuclear Location Signal (NLS)
family of proteins
associated with the
nuclear pore
complex
nuclear transport
receptors
Example of Nuclear transport receptors mentioned in the PPT
Importin and Exportin
recognize
the NLS and bring
proteins into the nucleus
Importin
Work in the opposite direction with importin. These recognize other signals
Exportin
__________ have no nucleus
Prokaryotes
The nucleus is About _______ of the cell volume
10%
Eukaryotes generally have 1 nucleus except
for ____, _______and ______________
rbcs, osteoclasts and skeletal muscles
-nucleus is the first organelle to be discovered
-First described by _______________
Franz Bauer in 1804
Overall Function of the Nucleus
-Storage of genetic material
-Controls cell growth and reproduction
-Home of essential cellular processes (i.e.
replication, transcription )
- Regulates passage of materials in and out of
nucleus (RNA, ions, nucleotides, aa etc)
The Nucleus contains __________ nuclear pores,
regulated by a protein structure,
the ____________.
-3000
-nuclear pore complex
The discovery of nucleus was presented by Scottish botanist, ___________
during the 1831 meeting of the Linnaean
Society of London
Robert Brown
In the nuclear pore __________ (<mw 20,000) can pass right through, _________ are strongly
regulated.
-small molecules
- larger molecules
The Interior of nuclear envelope is supported
by _______________ ( on the
nucleoplasm side)
nuclear lamina
A network of 10nm-diameter
protein fiber
Lamins
3 Functions of nuclear Lamina
(1)reinforces structure of the nucleus,
(2) anchors chromatin
(3) important in replication and transcription
*analogous to
cytoplasm
* Includes the soluble protein
nucleoplasmin
* Highly gelatinous, sticky
fluid that supports
chromosomes and nucleolus
* Suspension substance for
organelles inside nucleus
* Also dissolved are
nucleotides and enzyme
Nucleoplasm
Nucleoplasm includes soluble protein called?
nucleoplasmin
Functions of Chromatin
- Determines chromosome structure
- Changes chromosome structures associated with cell division
- Regulates chromosome function
In all cells of female
individuals, one of the two X
chromosomes is
permanently _________ and
________________________ in
transcription.
-inactivated
-does not participate
The inactive X chromosome is recognizable in epithelial cells from the oral cavity as a single granule called ___________________ that is attached to the nuclear lamina
sex chromatin (Barr body)
In _____________, the sex chromatin takes the form of a drum stick-like appendage to
the nucleus. These structures have been used
for gender testing
neutrophils
-25% of volume of nucleus
-Site of ribosome biogenesis
-Form around specific chromosome regions
known as NOR
Nucleolus
Main function of the Nucleolus
combine rRNA + protein to form
incomplete ribosome
Uninterrupted chain from nucleoplasm to
interior of nucleolus (nucleolar passages) allow
macromolecules up to ___________ to circulate
through organelle
2000 kd
Importance of Folded DNA Structures
a. for storage in the limited size of the nucleus
b. during mitosis and meiosis, it allows for neat
segregation into newly formed cells
c. manner of folding determines activity of
genes in a cell
All histone proteins have a large positive charge; between 20 and 30% of their sequences consist of the____________,
_____________and _____________
-basic amino acids,
-lysine
-arginine
Histones are large positive charge, meaning histones will bind very strongly to the _______________ in forming
chromatin.
negatively charged DNA
- At the pore, the inner and outer membranes come
together forming an opening. - It is lined with protein
Nuclear Pore
Nuclear Pores contains at least ________ proteins, including _____________, extending to the cytoplasm.
-100 Nucleoporins
-Cytoplasmic Filaments
Nuclear Pores regulate traffic into and
out of the nucleus by means of the
_________________
Nuclear Location Signal (NLS)
How do we know the function of NLS ?(Experiment 1)
modify the amino acid sequence (by site-directed-mutagenesis)
Modifying the amino acid sequence (by site-directed-mutagenesis). Process and conditions:
- “SV40 virus t-antigen” a protein molecule, M.W. 90,000
- normally enters the nucleus if injected into cytoplasm.
- normally accumulates in the nucleus (one-way)
- experimentally visualize the location of the protein
– using a fluorescent tag which glows
under UV light
– Set up a microscope with ultraviolet light
source - An experimental alteration of the sequence (pro-lys-thr-lys-arg-lys-val-) completely prevents accumulation in the nucleus
What happens when we use recombinant DNA techniques to add the NLS to a dummy protein?
Adding an NLS to a dummy protein using recombinant DNA techniques allows the protein to be transported into the nucleus of a cell, where it can perform its intended function.
Example of this is Normal or modified Bovine Serum Albumin (NLS added) and injected to the cytoplasm
Protein binds to a two-protein complex. Which are?
importin a (Alpha) and importin b (beta)
___________ is a receptor for the NLS portion of the protein (i.e. it recognizes and sticks to this region.)
Importin a (alpha)
Complex and protein stick to cytoplasmic filament is mediated by _____________
importin b (beta)
- Not an energy consuming step, it can go back at this point unless captured by the RanGTP in next step
Complex moves into nucleoplasm
importin b stays on the ____________ after binding and complex dissociation.
Ran-GTP
Ran-GTP - importin b complex moves back to the cytoplasm, ________________
down a concentration gradient
the Ran-GTP is converted to____________ by the enzyme ______. This causes it to loosen from importin b
- Ran-GDP + Pi
-RANGAP
an exportin molecule binds to ___________, setting it up for transport out of the nucleus
importin a
Enzyme _______ re-forms Ran-GDP to Ran-GTP
RCC1
Mechanism of Protein complex through nuclear pore complex
- Protein binds to a two-protein complex (importin a and importin b)
- Complex and protein stick to cytoplasmic filament
- Complex moves into nucleoplasm
- Complex binds to another protein (Ran-GTP)
- Importin b complex moves back to the cytoplasm, down a concentration gradient
- the Ran-GTP is converted to Ran-GDP + Pi by the enzyme RANGAP. This causes it to loosen from importin b
- an exportin molecule binds to importin a, setting it up for transport out of the nucleus
- Ran-GDP diffuses back to the nucleus
- Restoration to initial state
___________ is an energy source and controls
the process
GDP to GTP conversion
Molecules always diffuse from high to low concentration, so if the gradient is maintained, it can be used to __________________________
bring importin b back to the cytoplasm
RCC1 occurs only in the _________, RANGAP occurs in ___________
-nucleoplasm
-cytoplasm
By breaking down ________ and thereby removing it, RANGAP maintains the concentration gradient. It can take the
other molecule out with it.
Ran-GTP
By changing _________ back to Ran-GTP, RCC1 maintains the gradient helping _______ to diffuse back into the
nucleus
Ran-GDP
