WEEK 1 - NUCLEUS Flashcards
THE NUCLEUS
Eukaryotic def
An organism whose cells have a membrane-bound nucleus. All animals, plants, fungi and many unicellular organism are eukaryotic
Nucleus def
a membrane-bound organelle in eukaryotic cells containing DNA/genetic information and contains the cell’s chromosomes and genetic material, and acts as the cell’s control centre.
What is the nucleus
-contains DNA arranged in chromosomes
-surrounded by the nuclear envelope, a double nuclear membrane (outer and inner), which separates the nucleus from the cytoplasm to protect from damage of mechanical forces the cell may experience
-nuclear membrane is supported by a meshwork of intermediate filaments (nuclear lamins), help keep the nuclear structure
-Outer membrane is continuous with the rough endoplasmic reticulum
-nuclear envelope contains pores which control the movement of substances in and out of the nucleus
RNA is selectively transported into the cytoplasm, PROTEINS are selectively transported into the nucleus
RNA def
Ribonucleic acid, nucleic acid is present in all living cells. Its principal role is to act as a messenger carrying instructions from DNA for controlling the synthesis of proteins, although in some viruses RNA rather than DNA carries the genetic information
Why is the nucleus important
- Separates fragile chromosomes from cell contents – crucial for proper function of
cell as if DNA is damaged we may not get correct protein or any protein at all and organism likely to die - DNA replication, transcription and RNA processing - all in the nucleus
- Separates RNA transcription in the nucleus from translation machinery in the
cytoplasm - Nuclear envelope allows gene expression to be regulated
- mRNA undergoes post-transcriptional processing before moving from nucleus to
cytoplasm - control of gene expression at the level of transcription e.g. expression of some
eukaryotic genes controlled by regulated transport of transcription factors from
cytoplasm to nucleus
- mRNA undergoes post-transcriptional processing before moving from nucleus to
Cells with nucleus exceptions examples
red blood cells - have none
skeletal muscle cells - have several
most cells have a single nucleus
Nucleolus def
Largest structure in the nucleus of eukaryotic cells site of ribosome biogenesis, which is the synthesis of ribosomes and is also where ribosomal RNA genes are transcribed. Participates in the formation of signal recognition particles and plays a role in the cell’s response to stress
Nucleolus facts
- one or more nucleoli are found inside the nucleus
- most prominent (visible) in cells that are synthesising large amounts of protein
- sites at which ribosomes are assembled and ribosomal RNA is transcribed
ribosomes def
complex molecular machine that produce proteins from amino acids during protein synthesis or translation
Nuclear envelope
- Encloses DNA
- 2 concentric membranes (two or more membranes that have a common centre)-
penetrated by nuclear pore
complexes - Inner membrane contains
proteins that act as anchoring sites for chromatin and for the
nuclear lamina - inner and outer membrane continuous but maintain distinct protein compositions
- outer membrane – continuous
with ER and studded with
ribosomes - Proteins made are transported
into perinuclear space (space in-between inner and outer membrane) before coming out of nucleus
Chromatin def
a complex of DNA and protein found in eukaryotic cells. primary function is to package DNA molecules into more compact, denser structures.
Nuclear envelope during mitosis
- nuclear envelope dismantled so that microtubules can access the replicated chromosomes for segregation between the two daughter cells
- phosphorylation of lamins triggers disassembly of the nuclear lamina, initiating breakup of nuclear envelope
- NPCs disperse in cytosol (suspends the organelles) along with the nuclear membrane, as that releases the DNA in preparation for cell division.
- Some NPC proteins bound to nuclear import receptors – important in reassembly of NPCs at end of mitosis
- Nuclear envelope membrane proteins disperse throughout ER membrane
- Later in mitosis, nuclear envelope reassembles close to surface of chromosomes
Transport between the nucleus and cytosol
- some molecules are small enough to move between the cytosol and nucleus, however some can only go one direction
- nucelar basket - collection of material that need to be transported, found in inner membrane of NPC
Nuclear pores and NPCs
- Every NPC has roughly 30 different proteins (nucleoporins) and has eight fold rotational symmetry
- proteins make up Central portion of NPC which is orientated symmetrically, nuclear and cytosolic sides look identical
- nuclear porins make a mesh
nucleoporins
nucleoporins can be classified into
*transmembrane ring proteins that span the nuclear envelope and anchor the NPC to the envelope
* scaffold nucleoporins that form layered ring structures
* channel nucleoporins that line a central pore
channel nucleoporins
- many contain extensive unstructured regions, where the polypeptide chain is intrinsically disordered
- central pore filled with a high concentration of these disordered domains whose weak interactions form a gel that blocks passive diffusion of large molecules, contain large numbers of FG repeats
NPC key facts
- 3-4000 NPCs in typical mammalian cell
- ~ 1000 macromolecules/s in both directions simultaneously
- internal diameter ∼40 nm - large enough to accommodate ribosomal subunits, important as they need to go from the nucleus into the cytosol to finish off protein
- Pore filled with unstructured protein - numerous repeats of phenylalanine–glycine
(FG) motifs - weak affinity creates a gel-like mesh inside the NPC, gives orientation for proteins - Mesh - sieve - restricts diffusion of large macromolecules but smaller molecules pass
through- Small molecules (<5000 daltons) rapid diffusion = freely permeable
- Many cell proteins (40,000 daltons (∼5 nm diam) too large to diffuse passively
through the NPCs - different protein compositions in nucleus and cytosol - Mature cytosolic ribosomes (~30 nm diam) can’t diffuse through - protein synthesis confined to the cytosol
Regulation of transport through NPCs
- small proteins - continually shuttle between the nucleus and the cytosol
- larger proteins need import or export signal
- other proteins contain both nuclear localisation signals (NLS) and nuclear export signals (NES)
- relative rates of import and export determine steady-state localisation
- changing rate of import, export, or both - change the location of a protein
if you were to take out proteins from the nucleus and put them back into the cytosol, even the larger ones will reaccumulate back into the nucleus
Nuclear Localisation signals
Nuclear localisation signals (NLSs):
* responsible for the selectivity of active nuclear import process
* Most commonly - 1 or 2 short sequences rich in lysine and arginine (+ charged amino acids) - specific as sequence varies for different protein
* positively charged is so that they can interact with the importin in the nuclear pore complex.
* Located almost anywhere in the aa sequence - thought to form loops or patches on the protein surface
* As long as one of the protein subunits of a multicomponent complex displays a NLS,
entire complex will be imported into the nucleus
* NPC transport occurs through large, constitutively open, mesh-filled pore-
* fully folded proteins/ large multiprotein complexes can be transported in either
direction through the nuclear pore, in contrast to organelles protein translators of the ER, mitochondria and chloroplasts which has unidirectional transport and usually requires the protein to be extensively unfolded
Nuclear import receptors
- to initiate nuclear import, Nuclear localisation signals must be recognised by nuclear transport receptors
- Most receptors are karyopherins – importins or exportins
- each import receptor can bind and transport the subset of cargo proteins containing the appropriate NLS
- Receptors can use adaptor proteins that form a bridge between the import receptors and the NLS on the proteins to be transported, used for proteins that don’t quite fit
- Variety of import receptors and adaptors - cells recognise range of NLS
what if FG
phenylalanine and glycine
import receptors
- soluble cytosolic proteins
- contain multiple low-affinity binding sites for FG repeats found in the unstructured domains of several nucleoporins, is important because if the binding was stronger, the barrier would essentially become impenetrable.
- FG repeats - bind to transport receptors and their cargo, allowing transport complexes to move through the NPC and form a diffusion barrier that is selectively permeable to nuclear transport receptors.
- import receptors then bind the FG repeats that form the mesh inside the nuclear pore to disrupt
interactions between the repeats. - Receptor–cargo complex locally dissolves the gel-like mesh and can diffuse into and within the NPC pore
- import receptor then returns back to the cytosol for transport of the next cargo
interaction of nuclear import receptors with FG repeats
nuclear import receptors contain various low-affinity FG repeat-binding sites on they surface. This facilitates their initial recruitment to NPCs because of interactions with FG repeats found on the cytosolic fibrils of the NPCs. The interior of the NPC is filled with a mesh of FG repeat-containing proteins whose weak interactions with each other restrict nonspecific diffusion of proteins and other macromolecules through the pore, which also increases the rate of diffusion.
Proteins without surface FG repeat-binding sites cannot melt the mesh, and their diffusion through the NPC is comparatively slow
nuclear exports
- Export through NPCs depends on a selective transport system
- Relies on nuclear export signals on the macromolecules to be
exported - Export receptors bind to the export signal, either directly or via an
adaptor, and to NPC proteins to guide cargo to cytosol - Import and export transport systems similar but in opposite
directions:
the import receptors bind their cargo molecules in the cytosol, release them in the nucleus, and are then exported to the cytosol for reuse. export receptors function in the opposite direction - only difference is in nuclear exports cargo binding requires Ran-GTP (it promotes binding), while in importins cargo binding is mutually exclusive of Ran-GTP (promotes cargo dissociation)
Ran-G protein
present in both cytosol and nucleus required for the active transport of macromolecules into and out of the nucleus through nuclear pore complexes.
two types:
Ran-GTP - triphosphate
Ran-GDP - diphosphate
RAN proteins overview
- RAN is a small G protein (specialised proteins with the ability to bind the nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP)) that is essential for the translocation of RNA and proteins through the nuclear pore complex
- RAN is important for directionality (balance and homeostasis between nucleus and cytosol)
- the import of nuclear proteins through NPCs concentrates specific proteins in the nucleus and thereby increases order in the cell. the cell fuels this ordering by harnessing the energy of GTP hydrolysis by the GTPase RAN, which is required for both nuclear import and export.
- RAN in a molecular switch that can exist in two conformational states depending on wether GDP or GTP is bound
- these alternate guanine-nucleotide states of RAN are regulated by two classes of. partner proteins, which either promote the RanGTP or the RanGDP form
Ran GTPASE - directionality on nuclear import through NPCs
Cell fuels ordering process by harnessing the energy of GTP hydrolysis by the GTPase Ran, 2 conformational states, (depending whether GDP or GTP is bound)
- Ran GAP - found in cytosol and will cleave off a phosphate and produce Ran GTP (triggers GTP hydrolysis, GTP to GDP)
- Once it comes into the nucleus we have an addition of a phosphate and get GTP by Ran GEF (converts Ran GDP to GTP) which is located in the nucleus
- nucleus mainly contains Ran-GTP and the cytosol mainly contain Ran-GDP
- when an import receptor reaches the nuclear side of the pore complex, Ran-GTP binds to it and causes the receptor to release the cargo
Nuclear lamina
- Nuclear Lamina –
localised nuclear side of inner nuclear membrane - Meshwork of interconnected protein subunits (nuclear lamins)
- Lamins – intermediate filament proteins –
polymerise into 2D lattice. - Gives shape, structural support and stability to nuclear envelope
- Anchored by attachment to NPCs and integral
membrane proteins
*provides structural links between DNA, nuclear envelope and cytoskeleton - Interacts directly with chromatin, for example when cell division occurs it becomes important in the process of pulling chromosomal material apart (its sticky so help pulls)
- majority of lamins are found on the inner nuclear membrane
Nuclear lamin structure
- rope like structure with NH^2 on one end and COOH on the other
- formed from a monomer which is an alpha helix and coils to form a dimer (a molecule or molecular complex consisting of two identical molecules linked together), starts to strengthen
- staggered tetramer (a polymer comprising of four monomer units) of two coiled dimers, increases the strength
- 8 tetramers bind
- lipid-like anchor proteins (are proteins located on the surface of the cell membrane that are covalently attached to lipids embedded within the cell membrane) on COOH end attaches lamina to inner nuclear membrane
- Type V intermediate filaments are nuclear lamins, which are found in most eukaryotic cells and are part of the nuclear envelope. They are involved in the formation of the nuclear lamina.
DNA key points
- Deoxyribonucleic acid (DNA) - two long polynucleotide chains composed of four
types of nucleotide subunits (pentose sugar with a phosphate strand and nucleotide base) - Each chain is a “DNA strand”
- The two strands run antiparallel with hydrogen bonds between the base portions of the nucleotides
- A nucleotide is composed of a five-carbon sugar to which a phosphate group and a nitrogen-containing base are attached
- The nucleotides are covalently linked together in a chain through the sugars and phosphates, to form a “backbone” of alternating sugar–phosphate–sugar–
phosphate
Nucleotides
A nucleotide consists of a nitrogen containing base, a five-carbon sugar (ribose) and a phosphate group.
The phosphate makes the nucleotide negatively charged.
C-G forms 3 H bonds AND T(U)-A forms 2 H bonds
adenine + guanine = purine bases
cytosine + thymine + uracil = pyrimidine bases
what is the nucleoside
includes only the pentose sugar and nitrogenous base (not phosphate)
sugars
second carbon on pentose sugar will have no oxygen attached = deoxyribose (DNA), if OH group is present = ribose (RNA)
Nucleic acid polymers
- Nucleotides joined together by phosphodiester bonds between the 5’ and 3’
carbon atoms of adjacent sugar rings – forms polymer - Linear sequence of nucleotides - one-letter code e.g. AGCTT, starting with the 5’ end of the chain.
Creating the phosphodiester bond
condensation reaction- water is released
- The 5’ group of a nucleotide triphosphate is held close to the free 3’ hydroxyl group of a nucleotide chain.
- The 3’ hydroxyl group forms a bond to the phosphorus atom of the free nucleotide closest to the 5’ oxygen atom. Meanwhile, the bond between the first
phosphorus atom and the oxygen atom linking it to the next phosphate group breaks. - A new phosphodiester bond now joins the two
nucleotides. A pyrophosphate group (a phosphorus oxyanion that is made up of two phosphorus atoms linked by a P−O−P bond) has been liberated. - The pyrophosphate group is hydrolyzed (split by the
addition of water) which is catalysed by DNA polymerase, releasing a great deal of energy and
driving the reaction forward to completion.
Double helix
Nucleotides - covalently linked
Strands are held together by hydrogen bonds between base pairs
All bases the inside of the double alpha helix, sugar–phosphate backbones on the outside
The strands run anti-parallel
Base pairing
- Two-ring base (a purine) is paired with a single-ring base (a pyrimidine) - most energetically favourable
- Each base pair is of similar width - sugar–phosphate backbones a constant distance apart (equal width)
- bases can pair only if the two polynucleotide chains that contain the are antiparallel
the alpha helix
- energetically the alpha helix is a favourable structure
- Double stranded DNA molecule winds into a right-handed double helix –efficiency of base pairing
- One complete turn per 10.4 bp
- Two grooves, the major and minor groove
The chromosome
- Eukaryotes - 22 pairs of homologous (the same) chromosomes and 1 pair of non-homologous (X + Y)
- Exception - gametes (eggs and sperm) and some highly specialized cell types
- Chromosome - single long linear DNA molecule + proteins that fold the DNA into compact structure
- Chromosomes associated with other proteins (as well as numerous RNA molecules)
required for the processes of gene expression, DNA replication, and DNA repair - The complex of DNA and tightly bound protein = chromatin
- heterochromatin = area of highly condensed DNA and proteins
Karyotype
- a visual representation of an organism’s complete set of chromosomes, including their number, size, and shape.
centromeres and telomeres
CENTROMERES
* Attachment site for the two halves of each replicated chromosome (sister
chromatids)
* Not always in centre of chromosome, but is the waist line dent
* Keeps chromosomes properly aligned during cell division, keeps chromosomes together
* Centromere is important structure bc during replication chromosomes must be aligned or we don’t get appropriate cell division and cell could be lost/ or wrong proteins could be produced in the future
TELOMERES
* Repetitive stretches of DNA located at the ends of linear chromosomes
* Protect the ends of chromosomes to keep them from unravelling and being damaged – think
shoelaces
* In many types of cells, telomeres lose a bit of their DNA every time a cell
divides (happens as you get older). When all of the telomere DNA is gone, the cell cannot replicate and dies
* White blood cells and other rapidly dividing cells have an enzyme (Telomerase) that prevents their chromosomes from losing their telomeres – cells live longer
* Telomerase adds TTAGGG repeats to the ends of chromosomes, helps control the damage
* Role in cancer- chromosomes of malignant cells usually do not lose their
telomeres – fuels the uncontrolled growth
Cell cycle
INTERPHASE
* Cell actively expressing genes and synthesising proteins
* DNA is replicated – sister chromatids
M PHASE
* Occurs when DNA replication complete and the cell receives a signal that the DNA is good, nuclear division by mitosis occurs
* Nucleus divided into two daughter nuclei
* Chromosomes condense
* Nuclear envelope breaks down, mitotic spindle forms from microtubules and other proteins
* Condensed mitotic chromosomes captured by mitotic spindle and
complete set of chromosomes is pulled to each end of the cell
* separating the members of each daughter chromatid pair
* Nuclear envelope reforms around each chromosome set
* Cell divides into two daughter cells
* This phase is brief in mammalian cells (~1 hour) - rest of time spent
in interphase
DNA - size is important
- Most important function of DNA - form genes - including information about when, where and how much of each RNA molecule and protein is to be made.
- If the double helices of all 46 chromosomes in a human cell could be laid end to end, they would reach approximately 2 meters
- BUT the nucleus is ~6 μm in diameter.
- This is equivalent to packing 40 km (24 miles) of extremely fine thread into a
tennis ball. - DNA packaging uses specialized proteins that bind to the DNA and fold it - series
of organized coils and loops - DNA tightly compacted but remains accessible to the enzymes in the cell that
replicate it, repair it, and use its genes to produce RNA molecules and proteins
Chromatin - eukaryotes
- Chromatin (a complex of DNA, RNA, and proteins that forms chromosomes in the nucleus of a cell) - diffuse mass of DNA at interphase
What is interphase?
* period in cell cycle characterised by -
* G1 phase - cell undergoes growth,
* S phase - cell makes a copy of its DNA
* G2 phase - cell continues to grow, and prepares for cell division
Heterochromatin
chromatin regions that are
condensed during interphase and transcriptionally active
- Heterochromatin stains more densely than euchromatin
euchromatin
chromatin regions that are decondensed and DNA sequences are being transcribed into RNA
- Heterochromatin stains more densely than euchromatin (lighter part of nucleus)
Nucleosomes - eukaryotes
- Basic structural unit of DNA packaging - segment of DNA wound round histone proteins
- Fundamental subunit of chromatin
- Proteins binding to DNA to form chromosomes divided into two classes:
- Histones ( responsible for most basic level of chromosome packing, also known as nucleosome)
- Non-histone
- Complex of both classes of protein with nuclear DNA is known as chromatin
Histones
- histones are responsible for the first and most basic level of chromosome packing, a protein DNA complex called the nucleosomes
- if examined under a microscope when interphase nuclei are broken open, most of the chromatin contents appears to be in the form of a fibre
- if examined under a microscope where it is partially unfolded , the unfolded chromatin shows a DNA string with beads (nucleosome core particle – DNA wound round a
histone core)
Nucleosomes - content
*forming ‘beads-on-a-string’ with an area of linker DNA (DNA bond round the histone)
* Each nucleosome core particle - complex of eight histone proteins—two molecules each of histones H2A, H2B, H3, and H4—and double-stranded DNA that is 147 nucleotide pairs long
* This histone octamer forms a protein core around which the double-stranded DNA is wound
* 147 nucleotide pairs wraps 1.7 times around the histone core
* Each nucleosome separated from next by linker DNA (few nucleotide
up to about 80), gives flexibility
* Nucleosomes repeat at intervals of approx. 200 nucleotide pairs
structure of nucleosome core particle
the high-resolution structure of a nucleosome core particle reveals a disc-shape histone core around which the DNA is tightly wrapped in a left-handed coil of 1.7 turns. All 4 of the histones that make up the core of the nucleosome are relatively small proteins and they share a structural motif, known as the histone fold, formed from three helices connected by two loops. In assembling a nucleosome, the histone folds first bind to each other to form H3-H4 and H2A-H2B dimers
- H3 histone protein tail
NUCLEOSOMES PACKED INTO A CHROMATIN
FIBRE – THE ZIGZAG MODEL
- Chromatin in a living cell probably rarely adopts the “beads on a string” form
- Nucleosomes packed on top of each other – DNA more condensed
- Nucleosome to nucleosome linkages formed by histone tails, notably H4 tail
- ‘zig-zag’ effect makes them more compact and smaller
- histone H1is a linked histone, contacts both DNA and histone octamer and changes path of DNA as it exits from the nucleosome
looped domains
*loops fastened to form a ‘scaffold’ structure by proteins
* looped domains in interphase chromatin can unwind for gene expression/ DNA replication
What does the nuclear envelope consist of?
A single membrane plus the endoplasmic reticulum
A double layered membrane
A single unit membrane
A double unit membrane
What are benefits of having a separate, membrane bound nucleus?
Separation of delicate DNA from cytoskeletal filaments
Rapid response to the external environment
Enabled the evolution of split genes and alternative splicing (prokaryotes do not have non-coding DNA, which restricts their diversity and complexity because splicing enables production of multiple proteins from the same sequence)
What describes the structure of DNA?
A sugar phosphate backbone with protruding bases
Two anti-parallel strands
A single stranded molecule
What best describes the mechanism of DNA replication?
semi-conservative
What is the name given to the regions of DNA where replication begins?
Origin of replication, Replication Origin
How many chromosomes do we have?
46 diploid, 22 pairs
What are our remaining chromosomes called?
autosomes (any chromosome that is not a sex chromosome)
What are the two arms called on a chromosome?
short arm - p arm
long arm - q arm
What fold level of packaging does the beads-on-a-string form of chromatin cause?
3
What histones are found in the nucleosome core?
H1
What best describes the nucleosome core?
an octamer
How much DNA is wound around the histone core in the nucleosome?
146 nucleotides
Which additional protein is used to twist the beads-on-a-string form of chromatin into the 30nm fibre?
Histone H1
To what structure are chromatin loops attached?
nuclear scaffold
Approximately what is the maximum level of DNA packaging possible?
10,000
Which type of cytoskeletal element support the nuclear envelope?
nuclear lamina (intermediate filaments)
Do nuclear pores regulate the flow of all molecules into or out of the nucleus?
False - small non-polar molecules can pass through the pores in both directions. Movement of these small moiecules is not controlled by the pore
Proteins that are required in the nucleus must possess what?
nuclear import signal
Nuclear import receptors and nuclear export receptors may be called what?
importins and exportins
Tutorial map
DNA is wound around histones to from nucleosomes which is known as chromatin.
chromatin can be tightly packed (called heterochromatin) to inhibit transcription or loosely packed to enable transcription, known as euchromatin which stains lightly.
DNA is used as a template for transcription to generate RNA.
types of these include tRNA, rRNA and mRNA. rRNA is used to create ribosomes which are essential for translation to generate proteins.
What structures and functions do we have to protect our DNA?
- condensation of chromatin
- nuclear envelope with nuclear pores
- requirement of a nuclear localisation to enter the nucleus
- telomeres
the nuclear pore complex is made up sets of integral and peripheral proteins known as what?
nucleoporins
two types of membrane proteins that differ in how they are associated with a cell membrane. integral - permanently embedded, peripheral - loosely attached on inside or outside
in order to pass through a nuclear pore, a protein must contain a what?
nuclear localisation signal
which nuclear pore component forms the basket portion of the complex inside the nucleus?
fibrils
