201 Flashcards
birth of cell biology
1950s, ability to grow cells from many organs for a limited time however cancer cell lines grew immortally within the lab.
birth of cell bio (TEM)
development of new imaging techniques (TEM) allowed visualisation of cell structures, combined with staining techniques and now very advanced microscopy is now used.
Cell fractionation (birth of cell bio)
separates parts of the cell by size/density, rough er breaks down into smaller microsomes. The higher the speed the smaller the parts of the cell come out.
what could microsomes help study
the secretory pathway of a cell
The two main protein sorting pathways in eukaryotes
cytosolic proteins are sorted in the cytosol and membrane, luminal and secreted proteins are translated on the ribosomes of the rough ER
Where does translation start
on free ribosomes in the cytosol
where can proteins be targetted?
to the nucleus, to the mitochondria, a large amount of proteins are secreted out of the cell or trafficked to other organelles of the cell.
post translational translocation
chloroplasts translated in the cytosol and contains targeting sequence afterwards.
Co translational translocation
Membrane, luminal and secreted proteins are translated on the ribosomes of the rough ER:
How is a secretory protein targeted to the rough ER where it is needed to be translated?
What could be used to track secretory proteins and how they move throughout the cell? (OLD METHOD)
pulse-chase labelling system
pulse chase labelling system
label a subset of new proteins - pulse-chase: add a pulse of radiolabelled aa into cell culture media and chase it with normal aa. Activity can be followed using microscopic techniques (autoradiography).
Finding after pulse-chase labelling secretory proteins (observing trafficking)
new proteins very quickly associated (made) with the membrane of the ER -> ER important for secretion.
process of the pulse chase labelling system
- choose cell type that secretes proteins
- add a pulse of radiolabelled aa into cell culture media and chase it with normal aa
- follow where the radioactivity goes using microscopic techniques (autoradiography)
- isolate microsomes and treat with detergent and protease
What effect did the detergent have on the microsomes in the pulse chase labelling system
break down the fatty membrane of the microsome
what effect did protease have on the microsomes in the pulse chase labelling system
if detergent was added it would destroy the protein as the microsome membrane had been broken. If the membrane was not broken down the protein was protected.
what was the finding after adding protease and detergent to the pulse chase labelling system (microsomes)
found that proteins were translocating inside the lumen of the ER
How are proteins targeted to different parts of the cell? (SIGNAL HYPOTHJESIS)
Hypothesised that the secreted proteins should contain a signal telling where to direct them within the cell (signal hypothesis).
How could u explore the signal hypothesis?
choose and culture a cell type that secretes proteins, fractionate some membranes of rough ER, create cell free system for in vitro translation. Purified an immunoglobulin mRNA and translated it in vitro. Disrupted the membrane at different time points and examined the size of proteins.
The readout experiment
Investigating signal hypothesis:
- in vitro translation of IgG (immunoglobulin) mRNA
-disrupt microsomes with detergent
- translation finishes: Readout
- two sized polypeptide chains observed from one mRNA
Results of the readout experiment and the gel electrophoresis
two sized polypeptide chains observed from one mRNA, one longer precursor protein (if the microsomes were disrupted) which contained the signalling sequence and one mature sized shorter protein found in microsomes
what is the highly conserved signal sequence
N terminus start, positive charged amino acid then 6-12 hydrophobic amino acids.
experimental evidence for signal hypothesis
1) direct relationship between larger precursor protein and smaller mature protein
2) mature secretory protein was only produced if microsomes were present
3) mature secretory protein were protected from digestion in microsomes
4. conserved sequence sequenced.
How does the protein get through the ER (to the lumen)
through the sec 61 translocon
- conserved pathway/protein from yeast to humans
Structure of sec 61 translocon
pore with a plug that acts as a channel in the membrane, chain elongation at the ribosome sufficient to drive the polypeptide through the pore.
SEC 61 CO-TRANSLATIONAL TRANSLOCATION
translating and translocating at the same time as the polypeptide is made and being pushed through the membrane via translocon pore
co-translational translocation pathway (DETAIL) FOR SECRETORY PROTIEN
1) N’ signal sequence 6-12 aa sequence translated
2) Signal recognition protein binds to the signal sequence & ribosome and stops translation
3) SRP targets the complex to the ER by binding the SRP-receptor in the membrane, GTP binding
4) SRP subunits hydrolyse GTP, polypeptide transferred to sec 61 translocon.
5) polypeptide elongates and translocates via the channel, the signal sequence is cleaved.
6,7,8) polypeptide is in lumen, ribosome dissociates and the channel closes.
Type 1 transmembrane protein
has signal sequence, the N terminal, will be translated until it reaches a series of hydrophobic aa (stop transfer anchor sequence) which slips sideways during translation through the translocon and embeds itself in the membrane and the rest of the protein is translated left in the cytoplasm. N TERMINAL INSIDE LUMEN C TERMINAL OUTSIDE IN CYTOPLASM.
stop transfer anchor sequence
a series of hydrophobic amino acids in a TYPE 1 transmembrane protein slips sideways during translation through the translocon and embeds itself in the membrane
Topology of a protein
which end of the protein is on the inside or outside of the cell/ orientation or no. of times a polypeptide crosses a membrane
Type 2 transmembrane protein
no ER targeting sequence at the start of the protein, translated in the cytosol, N TERMINAL IN THE CYTOSOL. Also has a hydrophobic sequence called the signal transfer sequence which is recognised by the SRP and taken to the membrane to be inserted via the C terminus first.
signal transfer sequence
series of hydrophobic amino acids in TYPE 2 transmembrane proteins, this can be recognised by the signal recognition particle (SRP) which can be carried down to the translocon and inserted in the membrane.
GETTING TYPE 1 PROTEINS INTO THE MEMBRANE (TYPE 1 TRANSMEMBRANE PROTEIN PATHWAY_)
1) new polypeptide chain-ribosome complex associates with translocon, translated and the ss is cleaved
2) reaches stop transfer anchor sequence at the translocon
3) prevents further translation into lumen
4) stop transfer anchor moves laterally into the lumen, through cleft in translocon subunits
5) polypep anchored in membrane, translocon closes and synthesis finishes in the cytosol leaving C terminus.
6) ribosome dissociates leaving protein tethered in membrane
signal anchor
anchors protein into the membrane
post-translational translocation
protein is already made and folded and then transported somewhere else (yeast)
yeast (post-translational translocation) differences from co-translational translocation
pre folded in the cytosol, has to find its way to the ER by itself, has a signal sequence but no signal recognition particle to bind and bring it to a receptor.
yeast post translational translocation process
1) signal sequence enters translocon by direct interaction and slides through pore to the ER
2) ATP is hydrolysed from a chaperone Bi-ATP and ADP added to the protein chain
3-4) ADP stabilises the unfolded polypeptide chain and pulls it down into the ER lumen
5) ATP binding to BiP causes release, protein folds
6) signal sequence is cleaved off
Mitochondrial targeting
contain their own DNA but dont make all the proteins they need, conserved membrane translocation proteins with bacteria - post translational translocation
- pre proteins synthesised with an amphipathic alpha helix signal sequence
pre proteins synthesised with… (mitochondria)
amphipathic alpha helix signal sequence -> matrix targeting sequence
Mitochondrial post translational translocation process
like yeast, has no ribosome to push protein down and finds it way to membrane.
1) pre protein translated in cytosol with amphipathic SS, kept unfolded by chaperones Hsp70 + ATP.
2) SS binds to import receptor
3-4) receptor transfers pre protein to import pore
5) translocons of the outer and inner membrane line up, tom passes pre protein to tim complex
6) in matrix, Hsp70 binds and pulls pre protein inside, electrostatic gradient also provides energy
6-7) protein folds and SS is cleaved off
examples of post translational translocation
nucleus targeting, mitochondrial targeting and yeast.
Nucleus post translational translocation
Uses a nuclear localisation signal ( C’ sequence of 7 basic aa). Interacts with a nuclear transport receptor-importin and imports to the nucleus
WAYS OF STUDYING THE SECRETORY PATHWAY RER-> GOLGI
1) in vitro pulse chase radioactive aa and autoradiography of cells from secretory tissue r
2) in vitro live cell fluorescent fusion protein in cells
3) yeast conditional mutant: reversible temp sensitive mutation in yeast
Yeast conditional mutants: accumulation in the cytosol
defective function of the transport into the ER in the secretory pathway
Yeast conditional mutants: accumulation in the rough ER
Defective function of the budding vesicles from the rough ER
Yeast conditional mutants: Accumulation in ER-to-Golgi transport vesicles
defective function of transport vesicles with golgi
Yeast conditional mutants: accumulation in the golgi
defect in transport from the golgi to secretory vesicles
Yeast conditional mutants: accumulation in secretory vesicles
defective in transport from secretory vesicles to cell surface.
How are proteins transported out from the lumen of the ER in the secretory pathway?
vesicles that bud and fuse to from membranes carrying proteins, coat protein complexes.
how is budding initiated as proteins transported out from the lumen of the ER in the secretory pathway?
initiated by the polymerisation of coat protein complexes.
coat protein complexes process
budding initiated by coat protein complexes, COP interact with cytoplasmic tails (parts of transmembrane protein hanging outside in the cytosol) of membrane proteins and recruit cargo bound to membrane proteins within the lumen = cargo gathered into vesicle. Once away from the ER, they hydrolyse GTP and coat proteins come off leaving cytoplasmic tails. Vesicles movement thru cytosol via motor proteins. Fusion to target membrane occurs through SNARE binding
SNARE binding (cis-golgi/target)
v-SNARES (vesicle) bind to t-SNARES (target) and fuse with the golgi membrane. Proteins once in the vesicle will now be in the Cis Golgi.
How do coat protein complexes form? FORMATION OF COPII COATED VESICLES FROM THE ER
SAR1 BINDING PROTEIN exchanges GDP for GTP, SAR1 gets excited and changes conformation, inserting tail into membrane. Coat proteins will then bind to the energised Sar1-GTP and tails of membrane proteins.
Simplified COPII process
1) Sar1 exchanges GDP -> GTP: Sar1-GTP embeds in membrane and recruits coat proteins, selects cargo (membrane proteins).
2) Coat assembly around vesicle
3) GTP hydrolysis
4) uncoating
what coat proteins are used moving toward the golgi
COP II proteins
what allows fusion to the cis-golgi membrane?
v-SNARES tht bind to t-SNARES and fuse with the golgi membrane
what coat protein is in use from the cis-golgi back to the ER
COPI
how are cargo in vesicles transported ?
actively transported along microtubules (tubulin) via motors
what kind of transport is the vesicle transport to the ER or to the GOLGI
active transport along microtubules (tubulin) via motors
microtubule tracks
vesicles transported by motors, have a positive and negative end (near middle of the cell and pos near plasma membrane) as these run throughout the cell
The ER-Golgi intermediate compartment
mammalian cells have one but yeast doesnt
dynein (red)
retrograde motor (-) back to ER, backwards trafficking
kinesin (blue)
anterograde motor(+) forward trafficking moving forward in secretory pathway to plasma membrane.
Motor transport confusion
the proteins in the secretory pathway have to move from the ER to the golgi but the golgi is located more toward the centre of the cell so the proteins have to migrate retrograde via dynein first before heading back out via kinesin to outside of cell
Motor travel towards golgi or er first
toward the golgi via anterograde dynein to head toward centre of the cell from the ER (-) then move anterograde via kinesin (+) to the outside of the cell after the golgi (+), moving back before continuing forward.
bi-directional process)
COPII vesicles move anterograde or retrograde to the golgi from the ER?
anterograde transport FORWARDS (in the pathway) toward the golgi, via DYNEIN motor toward end of microtubule as the golgi is located centrally
what are COPII vesicles initiated by
SAR-1
how can proteins transported to the golgi be transported back to the ER?
via signals and COPI proteins via retrograde transport initiated by Arf GTP-binding protein recruiting coat proteins binding to cytoplasmic tails
COPI process
COPI protein vesicle transport initiated by Arf GTP-binding protein which recruits coat proteins that bind to cytoplasmic tails creating a COPI vesicle which will go from the golgi to the ER. This is a retrograde process via kinesin as it is backwards in the secretory pathway but forwards in the cell (+)
SELECTIVE RETRIEVAL PROCESS
SOLUBLE RESIDENT ER PROTEIN contains a KDEL sequence- protein will bind KDEL receptor tightly in the cis-golgi and will recruit COPI coat proteins and be transported back to the ER. When it will be released due to lower [H+] (higher pH) in the lumen.
SELECTIVE RETRIEVAL FUNCTION
recycles back essential ER proteins such as vSNARES, returns missorted resident ER proteins (BiP), retrieval sorting signal to direct ER cytosol or membrane, selective binding to receptor based on pH (more acidic golgi compared to ER)
retrieval of proteins missorted forward from the ER to the golgi. EG KDEL RETRIEVAL SEQ
Missorted protein makes way to golgi via COPII has a retrieval sequence, which can bind to receptor in the golgi -> recruited into coat protein. This is selective retrieval via COPI vesicles via GTP binding protein: ARF.
is KDEL for soluble or membrane proteins
SOLUBLE PROTEINS LIKE ENZYMES
Sorting signals
are structural features in proteins that control their targeting in/out of the cell
KDEL SEQUENCE
ROUGH ER RETENTION SIGNAL, DELETE THIS SEQ= PROTEIN SECRETION, ADD SEQ = PROTEIN retained in ER <- testing
Lys-Lys sequence
KKXX, for membrane proteins from the ER to be returned back such as vSNARES.
is GFP targeted to the ER?
no, its a cytosolic protein
ALL ER RETENTION SIGNALS:
KDEL: signal is SOLUBLE CARGO proteins found in the lumen - directs return to ER lumen via KDEL receptor binding with COPI (eg. soluble enzymes) and Lys-Lys (KKXX) sEQUENCE: membrane proteins from the ER can be returned back (vSNARES)
ER EXPORT SIGNAL
DI-ACIDIC sequence, directs membrane proteins and bound cargo forward to cell membrane, signals in cytoplasmic domain of membrane cargo proteins
Three membrane components of the golgi
cisternae
DI-ACIDIC SEQUENCE
ER EXPORT SIGNAL THAT DIRECTS MEMBRANE PROTEINS TOWARD CELL MEMBRANE
golgi cisternae
consisted of the trans medial and cis golgi
are proteins translocated through the cisternae?
no, they always remain in vesicles, vesicles merge together into the cis-golgi, they dont bud and fuse through they mature due to retrograde loss of material.
cisternae
three membrane components of the golgi, each differing in enzyme composition (glycosidases or glycotransferases).
What is the point of the ER/GOLGi
post translational modifications: making the proteins more stable and able to survive the hostile environment outside the cell.
cisternal maturation
the cis golgi matures into the medial golgi, a new cis golgi is formed via fusion of ER vesicles, the maturation process includes retrograde vesicular transport of resident golgi proteins (new formed medial golgi sends vesicles back and trans send it to the medial)
- proteins destined for secretion after modification are transported from the trans golgi to plasma membrane
- membranes fuse -> protein -> extracellular space
how do proteins transport through the golgi complex
vesicles dont bud they fuse through, they mature by retrograde loss of material and cisternae process into medial and trans golgi via cisternal maturation.
Retrograde vesicular transport
of resident golgi proteins from the newly formed medial golgi go to the cis golgi and trans golgi sends vesicles retrograde to medial golgi. The compartment identity changes as cis golgi eventually becomes trans golgi.
N-linked glycosylation
post translational modification- addition of sugars to polypeptide by resident RER enzymes to asparagine residues (N is aa code for asparagine)
Occurs during co translational translocation
Oligosaccharyl transferase recognises tripeptide sequence NH3+ …-Asn-X-(Ser/Thr)- COO N-X-S/T and adds complex sugar groups.
four main modifications of post translational modifications
- forming and assembly of multi subunit proteins which occurs in the ER
- disulfide bond formation (ER)
- glycosylation - carbs (Sugar) modifications (ER/GOLGI)
- specific proteolytic cleavages (ER/GOLGI/SECRETORY VESICLES)
what parts of post translational modifications occurs in the golgi
glycosylation and proteolytic cleavages which occurs in the ER, GOLGI and secretory vesicles
what is the point of post translational modifications
- quality control
- preparing protein for extracellular environment -> more vulnerable outside cell
- structural stability
- diversity of proteins - production of distinct molecules for signalling/communication
- activation/inactivation of enzymes ability
when does n linked glycosylation occur
occurs during co translational translocation to asparagine
what parts of post translational modifications occur in the ER
- forming and assembly of multi subunit proteins
- disulfide bond formation and
glycosylation that occurs in the ER and GOLGI
and proteolytic cleavages
ALL OF THEM
the sequence that oligosaccharyl recognises
tripeptide sequence of N-X-S/T and adds a complex sugar group the to the asparagine.
what happens as soon as the precursor oligosaccharide is added in n liked glycoslyation
immediately the cell starts trimming sugars off! precursor oligosaccharide processed and trimmed in the ER by glycosidases, if this is not trimmed to a core-glycan they wont be exported the the golgi making this a quality control step.
What happens if one of the asparagines were mutated?
No glycosylation, accumulation in ER lumen unfolded and will be degraded.
What is an example of a quality control step?
The N-linked glycosylation of asparagine and the trimming via glycosidase into the core-glycan to be exported to the golgi via COPII resulting in a highly modified protein
Clathrin-adaptor protein coated vesicles
vesicles that bud from the trans-golgi network (TGN) and plasma membrane during exocytosis have a two layered coat. Inner layer is adaptor protein complexes and outer layer is clathrin.
Clathrin-adaptor protein vesicles function and structure
adaptor proteins bind cytosolic domains of membrane proteins to determine what cargo is to be transported, clathrin polymerises to form lattice with intrinsic curvature and doesn’t associate with protein domains in cytosol.
-uncoating occurs through GTP hydrolysis by ARF helped by 2 accessory proteins
- clathrin-AP complexes transport to the lysosome and endocytosed cargo from the cell membrane.
how do clathrin-adaptor protein vesicles release from the membrane?
DYNAMIN: polymerises around the neck of the bud and hydrolyses GTP, stretches neck until it pinches off.
Dom-neg mutant of dynamin
= no vesicle budding. Scientist test for its function.
Dynamin
polymerises around the neck of the bud of the clathrin-adaptor protein coated vesicles and hydrolyses GTP, stretches neck until it pinches off.
lysosome
digestive and recycling compartments - break down macromolecules to monomer building blocks, organelles. acidic pH maintained by proton pumps, filled with digestive enzymes.
targeting proteins to the lysosome
lysosomal proteins transported through RER golgi, Mannose-6-phosphate modification in cis-golgi is the lysosomal sorting signal (N-acetylglucosamine phosphotransferase is essential)
M6P prevents processing into a secretory vesicle -> segregated at the trans golgi network
mannose-6-phosphate
lysosomal target signal from the glycosylation (modification) in cis golgi. M6P prevents processing into secretory vesicle and segregated at the trans golgi network. A SUGAR SIGNAL not aa. Trans golgi network membrane has a M6P receptor -> binds and recruits them into clathrin/AP1 coated vesicles for transport to the endosome-lysosome
endosome
intermediate sorting hub, more processing
M6P modified proteins head to lysosome
M6P-M6P receptor recruited into clathrin-AP1 coated vesicles -> endosome (processing) -> pH becomes more acidic as it goes -> endosome fuses into lysosome -> further processing of pro-protein in endosome -> functional protein-> recycling of receptors and protein coat
lysosomal storage disorders
battens disease, neurodegenerative brain diseases -> mutations in genes coding for lysosomal enzymes
- accumulation of lipids and waste in neurons
- incurable childhood disease
severe form of lysosomal disorder caused by
mutation in N-acetylglucosamine phosphotransferase = no M6P tag, protein is secreted.
what causes the lysosomal disorder
mutations in enzymes/for lysosome targeting ->missing lysosomal enzymes -> build up of proteins and lipids in vesicles and lysosomes.
M6P sorting signal found
by studying children with lysosomal disorder
how does endocytosis occur at the plasma membrane
via clathrin/AP2 coated pits that are spread across the surface of cells, endocytosed in a vesicle, delivery to different parts of the cell, degraded/recycled
endocytosis at plasma membrane
process of taking up fluid, particles or molecules from external medium
- encloses them in plasma membrane vesicles and internalises them -> take up of molecules (receptor mediated endocytosis)
receptor mediated endocytosis
the take up of molecules in endocytosis
what does endocytosis at the plasma membrane do?
- key regulatory step in determining protein composition in membranes
- used to ingest nutrients too large to go through a transporter
- can remove receptor proteins to down-regulate activity
endocytosis example molecules
low density lipoprotein, insulin (hormones), some glycoproteins, the iron carrying protein transferrin
enzyme replacement therapy
lysosomal storage disorder treatment -> endocytosis at plasma membrane
LDL (low density lipoprotein)
known as “bad” cholesterol and a major contributor to heart disease, research focus on how to reduce LDL in blood, why do levels get so high in the first place? -> discovery of LDL receptor
lipoproteins
package hundreds of lipid molecules into a large water soluble carrier for cells to take up from blood.
LDL receptor
low density lipoprotein taken up into cells, delivered to lysosome for breakdown/recycling
- LDL -> internalisation -> degradation
receptor mediated endocytosis of LDL
FAST PROCESS:
LDL binding receptor -> internalization -> degradation
1) LDL linked ferratin (Fe) binds its receptor in a pit
2)clathrin pit forms vesicle
3) LDL inside clathrin vesicle
4) LDL inside early endosome in 6 mins
receptor mediated endocytosis of LDL : INTERNALISATION
internalization is dependent on Asn-Pro-X-Tyr (NPXY) on the cytosolic segment of the receptor, bound by AP2 clathrin vesicle target to the endosome.
Dissociation from receptor via pH change (recycling 10-20 mins)
DEGRADATION NEXT IN LYSOSOME
LDL binding receptor -> internalization -> degradation
internalization is dependent on Asn-Pro-X-Tyr (NPXY) on the cytosolic segment of the receptor, bound by AP2 clathrin vesicle target to the endosome.
Dissociation from receptor via pH change (recycling 10-20 mins)
DEGRADATION NEXT IN LYSOSOME:
-lysosomes break down lipoprotein complex
- amino acids, fatty acids and cholesterol
- can be used elsewhere such as cell membrane synthesis
mutations that can cause familial hyper cholesterolemia`
LRLR- which parts? Mutation of NPXY sequence of LDL-receptor, mutation in ligand binding arm of LDL-receptor, AP2, genes that control internalization of LD2
= 2-6x higher LDL in blood -> development of heart disease
first trial of base editing in humans - lowering cholesterol
enzyme PCSK9 causes endocytosis of LDL receptors, what happens with LDL with fewer LDL-R?
- injection of treatment causes loss of function of PCSK9 in the liver -> what effect on blood LDL?
- adverse events did occur
- NZ human gene editing trial, base editing changes one base in gene -> switches of protein production in liver.
exocytosis in plasma membrane two categories of secreted protein process
constitutional and regulated
what replaces the membrane as endocytosis occurs constantly?
constant exocytosis and making of proteins. Contributing lipids back to the plasma membrane
Does exocytosis use coat proteins?
no, transport in the trans golgi network here uses no coat proteins.
constitutive secretory vesicles
EXOCYTOSIS OF PLASMA MEMBRANE:
starts as a pre-pro protein
after ER SS cleavage and processing in golgi = pro-albumin
after processing in secretory vesicle
= mature albumin
regulated secretion example
insulin secretion into blood from pancreatic (EXOCYTOSIS OF PLASMA MEMBRANE)
constitutive secretory vesicles example
albumin in blood from liver cells (EXOCYTOSIS OF PLASMA MEMBRANE)
regulated secretion
(EXOCYTOSIS OF PLASMA MEMBRANE)
starts as pre-pro protein
after ER SS cleavage and processing in golgi = pro insulin
after processing in secretory vesicle = mature insulin
held at TGN UNTIL A SIGNAL COMES into the cells to tell the cell to transport to the plasma membrane and exocytosed
difference between regulated secretion compared to constitutional secretory vesicles of the exocytosis of the plasma membrane
for the regulated secretion, the example mature insulin is held at the trans golgi network (TG) until a signal comes into the cells to tell the cell to transport to the plasma membrane and exocytosed
plasma membrane
hydrophobic core prevents unassisted movement of water soluble substances
diffusion of plasma membrane
influenced by conc gradients and membrane potential (electrochemical gradient)
transmembrane proteins
maintains differences between extracellular fluid and cytosol
potassium and sodium in extracellular fluid
high sodium outside >15mM and low potassium
potassium and sodium in the cell
high potassium and less sodium 15 mM
channels (transport proteins)
facilitated transport (passive transport) diffusion of ions down a gradient
transporters (transport proteins)
can be uniporter or cotransporters
uniporter (transport proteins)
movement of single molecule down gradient = facilitated transport
co transporters (transport proteins)
can be symporter or antiporter
symporter and antiporter
COTRANSPORTERS: couple transport of two different molecules . One down and ne against gradients 2ndry active transport
antiporter
COTRANSPORTER: both against conc gradient, powering for other free energy change allows something to move against gradient
symporter
COTRANSPORTER _> using one molecule travelling down conc gradient to pull another against their gradient
commonalities between channels and transporters
made up of multiple membrane spanning proteins that assemble in lipid bilayer to form aqueous pore, open/close of pore may be regulated/gated. May occur via conformational change. Chemical energy (atp hydrolysis) can be coupled to movement of molecules against conc gradient for pumps
pumps (transport proteins)
hydrolyse ATP to transport ions against their gradients in ACTIVE TRANSPORT, couple energetically unfavourable reaction with a energetically favorable one - electrochem gradient or ATP hydrolysis
despite all the commonalities between channels and transporters (transport proteins) …
huge variation in expression in diff types of cells depending on function
aquaporin
water is taken across plasma membrane of cells through facilitated transport through aquAPORIN
is aquaporin conserved
yes all the way through bacteria, plants to humans (12 aquaporins in human family)
structure of aquaporin
4 subunits make a channel with hydrophilic pore -> allows single file movement of water molecules down pore.
what if aquaporins didn’t exist/ 4 subunits and hydrophillic pore
whenever salts moved into cells, water couldn’t follow to maintain a homeostatic conc
which cells have high protein exp of aquaporin?
cells of kidneys, to reabsorb water from urine for concentrate.
How is glucose transported
taken into plasma membrane of cells through facilitated transport via uniporter called GLUT1 -> undergoes two conformational states as glucose binding site changes
how many GLUT genes in humans`
14
GLUT1
glucose is taken into the plasma membrane via facilitated transport through this GLUT1 uniporter as it is a transmembrane channel. Changing conformational states allows the glucose to go down its conc gradient
GLUT1 transport kinetics
very efficient at transporting glucose -> low kM (high affinity) compared to GLUT2 therefore it transports sufficient glucose into cells even when glucose conc is low. Reaches half of vmax even when there isn’t much concentration
vmax
maximum transport rate, vmax achieved when conc gradient is large and each uniporter is working at maximal rate
the number of channels are limited therefore
there is a maximal rate of transport
km
affinity of a transporter for its substrate, the conc of substrate at which transport is half of vmax
low km
high affinity
GLUT1 affinity
has a high affinity to its substrate: at external conc of 1.5mM, half of the transporters have glucose bound to the outside
GLUT2 affinity
far higher than GLUT1, needing external conc of glucose to be 20mM, but allows the uniporter to act as a glucose sensor
Why is the cytosolic conc of glucose so low?
rapid phosphorylation to G6P allows constant importing and maintenance of the glucose conc gradient
what cells contain GLUT1 and GLUT4 for insulin triggered transport
muscle and adipose cells as they are storage sites for glucose and fats.
GLUT4
muscle and adipose cells
- insulin responsive and stored in vesicles tethered to the golgi
GLUT4 response to insulin
insulin binding activates signalling in the cell
- kinesin transport vesicles
- GLUT4 receptor inserted into plasma membrane to inc glucose uptake in cell
GLUT4 response when blood glucose/insulin drops
endocytosis of GLUT4 and transported to endosome for recycling
defects in GLUT4
type 2 diabetes, cells cannot take up glucose
NA+ glucose symporter
transport of glucose into cells even when conc outside the cell is low, transports 2 Na+ ions down its conc gradient and couples transport of glucose against the conc gradient
-neither substance can move on its own and release in free energy as ion moves down gradient powers system
where are Na+ glucose symporters
intestinal + kidney tubule epithelial cells.
Pumps
couples ATP hydrolysis with the movement of ions/molecules against conc gradients.
P class pump and V class
P class pumps
set up membrane potentials, pumping ions
- plasma membrane of higher eukaryotes (Na+/K+) pump and
sarcoplasmic reticulum membrane in muscle cells (Ca2+ pump)
muscle p class pump
sarcoplasmic reticulum membrane in muscle cells for contraction (Ca2+) pump
membrane p class pump
plasma membrane of higher eukaryotes (Na+/K+) pump
V class pumps
H+ pumps creating different acidities
endosomal and lysosomal membranes in animal cells
how can a lysosome be more acidic than the ER
V class pumps control pH inside the cell
V class pumps controlling pH example
Vacuole is plant equivalent of endosome, ATPase driven transfer of 2H+ against conc gradient balanced by facilitated diffusion of Cl- ions to maintain electrical neutrality
result in decrease in pH of lumen of the endosome/lysosome
more v class pumps in the cis-golgi than ER
P class pumps in plasma membrane structure and function
teramer a2b2 structure -> catalytic and regulatory subunits -> ionic comp of cytosol kept constant
K+ ion intracellular and extracellular conc
high inside cell low in blood
sodium ion intracellular and extracellular conc
low inside cell < compared to outside
chloride ion intracellular and extracellular conc
low inside the cell but high outside
Ca2+ ion intracellular and extracellular
low inside and high outside cell
Na+/K+ ATPase in plasma membrane transports..
1 x ATP transports 3 Na+ out and 2K+ inside both against their conc gradient this sets up membrane potential to have more post charge outside.
Na+/K+ ATPase in plasma membrane process
1.3 Na+ bind with ATP then atp hydrolysis ending up in conformational change, opening pump to outside of cell allowing K+ ions to bind and Na+ is released -> dephosphorylation and conformational change allowing K+ to move inside cell. Setting up membrane potential of a more pos extracellular compared to neg intracellular.
receptor mediated endocytosis of GLUT4
to the endosome for recycling when glucose in blood lowers/insulin lowers AP2 clathrin
is symporters moving molecules the same or different direction
the same direction but one up its gradient one down (Na+ down taking glucose up)
is antiporters moving molecules the same or different direction
different directions coupling one moving down gradient with one moving up
transcellular transport of glucose/aa into blood
net movement of dietary glucose, aa and Na+ into blood through intestinal epithelial cells from intestinal lumen via 2Na+/glucose symporter. Then to blood via GLUT2 and Na+K+ ATPAse
Blood components
high Na+ low K+
Cytosol of intestinal epithelial cells
High K+ low Na+
resting membrane potential
differential dist of charged ions on each side of a membrane = electrical potential across a membrnae
Example of membrane impermeable to Na+, K+ and Cl-
equal numbers of pos and neg ions on each side therefore no membrane potential (no Na+ K+ channel)
membrane permeable only to Na+
Na+ channels allow inward flow, as pos charge in cytosol inside ~+60mV
membrane permeable only to K+
K+ channels allow outward flow, more pos charge in outside cell than inside ~-60mV
Resting potassium channels
K+/Na+ ATPase pump and non-gated K+ channels interact to generate a membrane potential
Plasma membrane contains many open (NON gated) K+ channels (few Cl-, Na+ and Ca2+)
resting potassium channels, resting membrane potential amount
-60 to 70mV relative to the inside of the cell
What interact to generate membrane potential
K+/Na+ ATPase pumps and non-gated K+ channels interact to generate a membrane potential.
the plasma membrane contains many what… channels
non gated K+ channels (few Cl-, Na+, Ca2+).
Generation of action potentials
transient change in membrane potential across the plasma membrane from pos inside to neg. Explosive entry of Na+ ions leading to depolarisation (less negative)
depolarisation
less negative, explosive entry of Na+ ions into the cell as -ve inside to +ve.
propagation of an action potential (direction)
AP can only propagate/travel thru cell in 1 direction at one time as sodium channels that are open to continue firing are ones further down axon.
Are Na+ channels normally closed or open
Na+ wants to flow inward down its conc and electrical gradient but normally Na+ cells are closed
Propagation of an action potential (PROCESS SUMMARY)
Change in voltage -> stimulate opening of Na+ voltage gated channels and explosive entry of Na+ into the cell as action potential causing pos change in cell and changes conc gradient. Resulting change in voltage triggers further opening of voltage gated Na+ channels down the axon.
-> triggers voltage gated K+ channels allowing more outward flow of K+ ions down conc gradient
-> return to baseline membrane potential (repolarisation)
The propagation of an action potential requires proper regulation
if not, we get hyperactive cells, causing AP to continue to fire (epilepsy) or cells not firing properly (understimulation.
Why is the propagation of AP unidirectional?
AP travels in one direction because these voltage gated Na+ channels become inactive after stimulation (channel-inactivating segment), plugging the pore to allow the cell to start repolarisation → back to normal membrane potential
Channelrhodopsin
light sensitive Na+ channel that allowed for Na+ inflow and AP for algae to move in response to light
optogenetics
using light activated channels to control cell function through manipulating membrane potential.
optogenetics uses
could insert into cells through DNA transection and could create transgenic mice with channel rhodopsins expressed in certain neurons
optogenetics in terms of study
used to study behaviours (thirst), brain conditions: parkinsons disease, epilepsy and PTSD.
TRPV1 cation channel
(Na+ and K+) opens in response to heat or cold and results in an action potential that causes burning sens and pain
how was TRPV1 found
cation channel responds to heat/cold: searched for genes responsive to heat or cold by treating sensory neurons with capsaicin -> watched for action potential
PIEZO2 (pressure)
mechanosensitive ion channel for touch and proprioception. 38 transmembrane helix topology.
How as PIEZO2 found
i.e. pressure mechanosensitive ion channel for touch and proprioception -> found by mechanically poking cells and watched for action potentials.
Yellow light sensitive channels
Cl- channels which stop action potentials firing in optogenetics.
Blue light sensitive channels
Na+ light activating channels in algae that causes algae to swim toward/away from light
What do nerve terminals contain
many vesicles that contain chemical messengers for secretion (neurotransmitters)
examples of neurotransmitters (chem messengers)
dopamine, serotonin, acetylcholine and GABA found in vesicles at nerve terminals
where are chem messengers/neurotransmitters made
they are synthesised from aa in the cytosol
arrival of an action potential in axon of presynaptic cell
causes fusion of vesicles to plasma membrane -> voltage sensitive Ca2+ channels in plasma membrane. Exocytosis of neurotransmitters from vesicles to synaptic cleft -> binding to receptors in post synaptic cell (muscle/neuron) which receives the chem signal.
where are the receptors for the neurotransmitters
on the post synaptic cell
neuromuscular junction neurotransmitter
acetylcholine
Neuromuscular junction AP process
volt gated Ca2+ channel opens -> Ca2+ inflow causes ACh vesicle fusion/exocytosis
ACh binds to ligand gated receptor -> Na+ in K+ out.
-> Localised depolarisation causes opening of V gated Na+ channel -> propagation of signal to V gates Ca2+ channel that signals SR to release Ca2+ into cytosol
what causes muscle contraction
Propagation of signal to voltage gates Ca2+ channel that signals SR to release Ca2+ into cytosol that causes muscle contraction (AP in Neuromuscular junction)
Botulism toxin (BOTOX!) inhibits …?
vesicle fusion with membranes -> prevent exocytosis of neurotransmitters (ACh) at neuro-muscular junction.
what does botulinum toxin do to ACh fusion with the PM
- binds to Ach motor neurons via receptor mediated endocytosis to endosome. One of the domains creates pore in vesicle on the way to the endosome and releases some of the protein (catalytic domain) into the cytosol of cell.
- This protease cleaves the v-SNARE on vesicles which prevents ACh from fusing with the plasma membrane
- this means no ACh → no muscle contraction as it causes paralysis
is botox permanent
no, botulinum toxin can be degraded by the cell and v-SNARES can be replaced. Making botox temporary.
uptake of neurotransmitters by cooperation of channels
neurotransmitters are taken back up by symporters back into the cell coupled with Na+ and Cl- (uphill transport) back via synaptic cleft to recycle the neurotransmitters
how do neurotransmitters get loaded into vesicles
via the action of V class H+ pumps coupled with an H+ antiporter that moves neurotransmitters in with the favourable movement of H+ exiting vesicle.
vGLUT
antiporter for Glutamate to enter vesicle
vGABA
antiporter for GABA to enter vesicle
vMAT
antiporter for dopamine and serotonin to enter vesicle.
neurotransmitter vesicles at synpase (BEFORE ARRIVAL OF AP)
- in vesicle (V class Pump and antiporter)
- trafficked to PM
- V and t snares bind to form SNARE complex -> docks vesicle at membrane (waiting for signal)
synaptotagmin
calcium sensor and allows the fusion of the vesicle and target snares -> neurotransmitter fuses -> released via exocytosis
Arrival of action potential down synapse steps following
opening of voltage sensitive Ca2+ channels -> Ca2+ detected by synaptotagmin which interacts with SNARE complex -> fusion with PM -> release
5. recycling
recycling of neurotransmitters and protein clathrin coat
Na+/neurotransmitter symporter reuptake neurotransmitter to be recycled and Clathrin/AP2 -> binds to signals and cytoplasmic tails that are pinched off by dynamin are endocytosed
Dopamine symporter
uptake of dopamine back into the cytosol
many drugs like to target symp
symporters
Cocaine/amphetamine and ritalin on DOPAMINE SYMPORTER
binds and inhibits/competes for the transporters for dopamine. prevents binding of dopamine → cant be taken up into cell and stays in synaptic cleft between neurones → continually signal to post synaptic cell
changing DAT trafficking and plasma membrane expression (inc signaling and continual stimulation on post synaptic receptors
Serotonin symporter
takes up serotonin into the cell using Na+, Cl- and K+ gradient
drugs that target serotonin symporter
antidepressants (fluoxetine, paroxetine) that act on serotonin reuptake transporter
drugs that target dopamine symporter
cocaine, ritalin and amphetamines
Antidepressants fluoxetine, paroxetine acting on serotonin symporter
Selective serotonine-reuptake inhibitors, inhibits reuptake of neurotransmitter = more serotonin in synaptic cleft and prolonged stimulation. More signaling to post synaptic receptors.
GABA transporters
transport GABA (inhibitory neurotransmitter) -> stops signaling.
antiepileptic drug
inhibits GABA uptake (more inhibitory neurotransmitters in cleft) -> prolonged suppression of signaling
Lorazepan
anti anxiety drugs -> target GABA receptors, binds to GABA-A receptor which is a ligand gated Cl- channel. Enhances binding of the neurotransmitter to receptor -> inc influx of Cl- into neuron. More neg charge in cell -> unable to fire action potentials = calming/sedative effect.
1928 griffiths experiment
the transforming principle, rough and heat treated smooth bacteria did not kill mice, smooth bacteria did. However when combining rough and heat treated smooth bacteria, the mice had died. This indicates a transforming component of smooth bacteria that changed the phenotype of the rough bacteria.
1944 avery-mcCarty-mcleod experiments
wanted to find the transforming component discovered to be present in 1928 griffiths experiment. Isolated DNA and found that DNA was the transforming component, soldified by removing DNA to test the hypothesis. A DNA virulence factor was changing the phenotype of the rough strain.
process of finding the transforming component (experiment one of 1944 avery-mccarthy-mcleod experiments)
fractionated heat killed smooth strain bacterial cells:
1. removed polysaccharides (carbs) enzymatically
2. removed protein using chloroform precipitation
3. used alcohol to precipitate the rest
4. obtained fibrous material -> when mixed with rough virulent strain transformed into virulent
how did we find out the transforming component was DNA based of the 1944 avery-mccarthy-mcleod experiments?
Elemental analysis of the fiborous component was consistent with DNA, so they did a second experiment by destorying the DNA with DNAse and following with RNAse etc. Only the DNA one survived after removal of DNA -> therefore DNA is the transforming component taken up by the rough strain from the dead smooth strain
what is the transforming component?
DNA-> the transforming component that was taken up by the rough bacterial strain from the death smooth strain. A DNA virulence factor is changing the phenotype of the rough strain.
INHERITANCE MUST COME FROM DNA -> this was unknown.
Introducing DNA into cells/organisms: transforming
changing bacteria
Introducing DNA into cells/organisms: transfecting
using plasmid vectors
Introducing DNA into cells/organisms: transducing
using viral vectors
photo 51
X-ray crystallography image of DNA -> barrel of the double helix
1952 DNA: race to identify structure
X ray diffraction images and ‘first parity’ rule helped determine DNA structure -> photo 51. Roaslind franlin and maurice wilkins discovered photo 51 taken by watson and crick.
Purines
2 rings, adenine and Guanine
pyrmidines
1 ring, thymine which is swapped out for uracil in RNA, and cytosine.
1953: Watson-Crick base pairing
important features:
ractions of A:T and GC are approx equal (first parity rule)
A-T has 2 hydrogen bonds and G-C has 3 (stronger)
first parity rule
ratios of AT and GC approx equal
IS A-T stronger or G-C
G-C because it has three hydrogen bonds whereas A-T only has 2.
1953 DNA double helix
proposed by watson and crick
-> backbone alternating deoxyribose sugars and phosphate groups (neg charge)
bases are purines (AG) or pyrimidines (TC)
directional antiparallel 5’ and 3’ ends to form supercoil
1953: DNA double helix major grooves
rich in chemical information, proteins can read the base pairs by identifying their H bonding properties down the major groove, includes DNA binding proteins- transcription factors, restriction enzymes (ecoRI), other intercalating agents important for cancer and experimentation (DAPI)
multi enzyme complex
In DNA replication, this complex is responsible for unwinding the DNA double helix, synthesizing new DNA strands, and ensuring the process is accurate. Duplicates each strand
DNA replication steps (simple)
- initiation, unwinding, separate and priming sites, little prime is start with RNA and add DNA onto those
- elongation: add dNTPs
- termination by have origins of replication converge on one another
Semiconservative replication
the process where DNA replication produces two DNA molecules, each containing one original strand and one newly synthesized strand, ensuring genetic information is accurately passed down
features of DNA replication
- multi enzyme complexes
- multiple start points
- semi conservative.
DNA organisation in the nucleus
DNA associates with histone proteins and forms nucleosome ‘beads’ in the nucleus
euchromatin
decondensed/relaxed chromatin associated with transcription as as they are not as tightly bound together like heterochromatin.
Transcription factors will access euchromatin much easier to transcribe RNA.
heterochromatin
associated with gene repression
1958 central dogma
Central dogma: purpose of DNA was to make RNA and the purpose of RNA was to make proteins → one way flow of info in cell.
Crick believed that proteins are the most important biomolecule
1958 sequence hypothesis
this hypothesis unites several remarkable pairs of generalisations
- the central biochemical importance of proteins and the dominating role of genes and in particular of their nucleic acid.
- the linearity of protein molecules and the genetic linearity within a functional gene
- the simplicity of the composition of protein molecules and simplicity of nucleic acids
DNA as heredity material, RNA as the message and PROTEIN as the machine.
1958 what did crick propose
that the purpose of DNA was to make RNA to make proteins in a one way flow of information in the cell
three keys of the sequence hypothesis
1958, the IMPORTANCE of proteins
the LINEARITY of protein molecules and the SIMPLICITY of the compostion of protein molecules.
what did crick not take into account with his 1958 central dogma and sequence hypothesis
didnt know or understand the fact that DNA can replicate itself as well as RNA, and the process isnt necessarily linear (information can go in different directions)
DNA Damage
chemical modification of bases, UV, reactive oxygen species, cosmic radiation and errors in DNA rep
errors in DNA replication
-> DNA damage.
Est. human mutation rate 2.5 x 10^-8 mutations/nucleotide site generation. ~70-150 base changes per gen
tobacco smoke on DNA
chemical mutagen/carcinogen, major component of tobacco smoke. INTERCALATES DNA and distorts double helix -> add extra chemical material onto guanine
UV exposure ot NDA
physical mutagen, dimerization of adjacent thymine bases in response to UV exposure. Thymine dimer causes ‘bulge’ in double helix.
DNA mutations
changes to the DNA code, we have a change to the DNA that is now encoded and embedded in there.
mutation types and consequences
synonymous: doesnt change amino acid
missense: changes aa
nonsense: changes aa to STOP
frameshift: (insert/delete) changes reading frame
Common nucelotide mutations (Cytosine)
Spontaneous deamination of cytosine as amine group replaced by water changes C to U and deamination of methylcytosine to thymine
spontaneous deamination of cytosine
Common nucleotide mutation: Amine group is replaced by water and changes cytosine to a uracil. Acts as a signal to correct the DNA strand as it uracil is a component of RNA.
Deamination of cytosine
common nucleotide mutation: Methylation is used for transcriptional regulation, methyl-cytosine goes through deamination, if we lose the amine here we end up with a thymine. Problematic and mutation would remain after replication. `
how does the DNA polymerase proof read enzymes
5’-3’ polymerase activity
3’-5’ exonuclease activity (backwards) distorted DNA strand moves into the exo site for correction as it is being replicated
Correction: proofreading enzymes (DNA POLYMERASE)
during DNA replication, incorrect base pairing will distort the DNA structure, DNA polymerases are proof reading(correct as they go)
MMR
DNA repair: mismatch repair
important factors to consider when thinking about DNA repair
mutation must be RECOGNISED, REMOVED and REPAIRED
BER
DNA repair: base excision repair
NER
nucleotide excision repair (DNA repair)
what does base excision repair do
repairs deaminated cytosines and oxidation products (the most common mutations) Specifically recognises thymine bases where cytosine should be. Cuts out incorrect base and repairs it.
Endonucleases
goes within the DNA sequence, doesn’t require being on the end of a nucleic acid and trimming back (cut anywhere in the middle)
APEI endonucelases
cleaves the abasic site in base excision repair after DNA glycosylase removes incorrect base
DNA glycoslyase in base excision repair
specific for incorrect/modified base, removes base leaving abasic site
DNA polymerase B and DNA ligase in base excision repair
removes backbone and replaces nucleotide after DNA glycosylase removes incorrect base and APEI endonuclease cleaves the abasic site. DNA ligase seals the DNA.
what does mismatch repair do
repairs small mismatches and slippage of repeated DNA and removes them/corrects it
mismatch repair
repairs small mismatches and slippage of repeated DNA
MutS: MSH enzymes recognise mismatch thru bulge in DNA structure, MUTL: MLH endonuclease nicks the damaged strand and DNA exonuclease removes segment containing mismatch. DNA polymerase omega repairs the gap and DNA ligase seals.
molecules involved in mismatch repair
MSH enzymes (recognise mismatch)
MLH endonuclease: cuts damaged strand
DNA exonuclease: removes segement
DNA polymerase omega and DNA ligase
what is the difference between mismatch repair and base excision repair
BSR removes one singular base to leave an abasic site for a singular common mutation whereas MR just removes an entire segment for fixing mismatches in DNA
MSH enzymes in mismatch repair
recognise mismatch through the bulge in DNA structure`
what repairs deaminated cytosines and oxidation products
base excision repair
Base excision repair
repairs deaminated cytosines and oxidation products, (C->T) 1. DNA glycosylase specific for incorrect/modified base removes base leaving an abasic site
2. APEI endonuclease cleaves abasic site
3. DNA polymerase B removes backbone and replaces nucleotide, DNA ligase seals
DNA ligase seals
the DNA in repair and replication
MLH endonuclease in mismatch repair and DNA exonuclease
nicks the damaged strand, DNA exonuclease removes segment containing mismatch
DNA polymerase omega and DNA ligase seals
DNA polymerase repairs the gap and DNA ligase seals it.
Difference between nucleotide excision repair and BNR, MR.
the lesion can be larger, for larger DNA errors with two different pathways (Global genomic repair NER or transcription coupled NER)
Nucleotide excision repair
repairs larger DNA errors/lesions, cuts out a larger region than base excision repair and has two pathways.
what are the two pathways of nucleotide excision repair
Global genomic repair NER and Transcription coupled NER
Global genomic repair NER (nucleotide excision repair)
corrects broad range of lesions (XPC + RAD23B enzymes recognize helix distortion)
XPC and RAD23B ezymes
Recognises helix distortion in nucleotide excision repair for global genomic repair NER.
Transcription coupled NER (nucleotide excision repair)
stalled transcription by RNA polymerase acts as a signal
Nucleotide excision repair process
either global genomic repair NER or transcription coupled NER: TFIIH (helicase): general trascription factor involved in transcription opens strands
RPA: ssDNA binding protein recruited to protect bubble as XP endonucleases cut damaged strand (24-32)
DNA polymerase replaces bases, DNA ligase seals.
how many base pairs excised by nucleotide excision repair
24-32
TFIIH (HELICASE) in NER
general transcription factor involved in transcription and is used to open strands in NER
How are DSBs repaired (double stranded DNA breaks)
either by non homologous end joining or homologous recombination repair
DSBS (double stranded DNA breaks)
replication fork collapse during DNA replication, ionizing radiation (chemical damage), DSBs are common; 5-10% cells in culture have a DSB.
in repairing DSBs which process is error prone?
non homologous end joining as they just stick both ends of the DNA without caring about which base pairs match and at the same time we lose base pairs.
Non-homologous end joining
occurs throughout cell cycle, DNA-PK+KU protein complex binds DSB ends, artemis (5’-3’ exonuclease) trims ends and DNA ligase joins ends back together. PRONE TO ERROR
what binds the DSB ends in non homologous end joining
DNA-PK + KU protein complex binds DSB ends for Artemis (5’-3’ exonuclease) trims ends
homologous recomb repair process
EXO1/DNA 2 exonucleases trim DSBs to create complementary overhangs, RAD51 helps overhanging strand to invade complementary strand, DNA polymerase extends invaded strand and displaces dark blue strand which also pairs with complement (Interlocked). DNA polymerase extends both strands and ligase seals. RESOLVASES cut holliday junctions (crossover structures) and ligases join ends.
Homologous recombination repair
uses homologous recombination to accurately repair break, uses other chromosome as accurate source of genetic info and only occurs during cell (S and G2) when chromosomes are located next to each other and available to use as template
when does homologous recombination occur
during cell cycle (S and G2 phase) when chromosomes are located next to each other and available to use as a template
when does non homologous end joining occur
occurs throughout the cell cycle
RESOLVASES in homologous recombination repair
cut holliday junctions (Crossover structures of dna and complementary strands) and ligases join ends.
what DNA repair problem results in Huntingtons disease
Mismatch repair, repeats of CAG in Htt gene can slip during replication or transcription causing loops/bulges. The wrong strand is removed resulting in using the loop.bulge as template and introduces extra repeats into the repaired strands. Too many glutamines in Htt protein.
what helps the overhanging strand to invade complementary strand in homologous recombination repair
Rad51
gene editing nobel prize
CRISPR-Cas9: deliberately introduce mutations or change base pairs in the genome 2020 nobel prize: Jennifer DOudna and emmanuelle charpentier
too many glutamines in Htt protein
causes huntingtons disease as MMR fails by using loop/bulge as template and introduces extra repeats into the repaired strand (removal of wrong strand by exonuclease).
jennifer doudna and emmanuelle charpentier 2020
nobel prize for CRISPR-Cas9 -> deliberately introduce mutations or change base pairs in the genome
CRISPR-Cas9
Clustered regularly interspaced short palindromic repeats, originated as bacteria defence against viral pathogens. The bacterial ‘adaptive immune system’ (compare to our antibodies)
CRISPR- Cas9 in bacteria
Bacterial Cas-9-tracrRNA-crRNA complex finds and cuts viral DNA (must include PAM- protospacer adjacent motif)
CRISPR- Cas9 in bacteria key elements
Cas9- enzyme that cleaves DNA
Tracer RNA and crRNA
PAM sequence
CRISPR-Cas9 as a tool
can turn the bacterial system into a gene editing tool, crRNA and tracrRNA linked to make a single guideRNA then the CRISPR-Cas9 can be encoded to cleave any sequence off.
How can we use CRISPR-Cas9 as a tool?
deliver Cas9 protein and guideRNA into the cell, or deliver plasmid/viral DNA vector or mRNA into the cell!
What happens after CRISPR-Cas9 cut?
Creates a DSB which we can use in aims of creating a disruptive error in non homologous end joining (frameshift and inactivation of protein) or homology-directed repair (similar to HRR) USING A DNA TEMPLATE WITH DESIRED SEQUENCE
BENEFITS OF USING CRISPR-Cs9 AS A TOOL FOR GENE EDITING
We can find a specific part of DNA we want to change by cutting off sequence we dont want and replacing it with a desired sequence with homology-directed repair
homology-directed repair
similar to homologous recombination repair, however we can use a DNA template with a desired sequence with CRISPR-Cas9.
Case study: gene editing for HIV prevention
2018 He jiankui CCR5 gene editing: first genetically engineered human babies from HIV pos mother: 1 baby heterozygous (CCr5 editing with no benefit) 1 baby homozygous (CCR5 editing). Off target effects unknowing, germline (heritable). Mosaicism.
ccr5 mutation
some people of northern european descent naturally carry CCR5 mutation (cant be recognised by HIV): ~10% heterozygous, 1% homo
1% naturally resistant to HIV infection.
nov 2018 he jiankui: ccr5
CCr5 gene editing in mouse, monkey and human embryos using CRISPR-Cas9 -> created first genetically engineered human babies (HIV pos mother)
Modified Cas9 (base editing) no double strand break
converts bases at a position indicated by the guide, uses mutant or dead Cas9 fused to cytidine deaminase (CBE) or adenosine deaminase (ABE)
traditional CRISPR editing is good for
good for introducing indels and small inserts/changes which requires a double stranded break (not really ideal)
modified Cas9 base editing vs traditional CRISPR editing
original good for introducing indels and small inserts/changes requiring DSB but not really ideal when most diseases are caused by C>T mutations: converts bases at a position indicated by the guide with no DSBs
case study: gene editing for cholesterol
High LDL cholesterol associated with coronary artery/cardiovascular diseases and death: VERVE101: mRNA in lipid nanoparticle, intravenous infusion -> edits A-T to G-C within a splice donor site and silences PCSK9 expression in hepatocytes -> LOWER LDL
housekeeping RNA
for ongoing cellular processes (rRNA/tRNA; transcription translation)
regulatory RNAS
non-coding, for splicing and control of gene exp.
tRNAs and small nuclear RNA made by which polymerase
RNA polymerase III
rRNA is made by which polymerase
RNA polymerase I and nucleolus
how many mRNA
~2% highly regualted, coding and made by RNA polymerase II
how many transfer RNA and rRNA
~96%
how many other RNAs (small nucleolar and micro RNAs)
~2%
Three RNA polymerase enzymes
POL II, POL III and POL I
POLYMERASE II
makes mRNA and snRNA: highly regulated process to give cells their identity and allow cellular functions
POL III
constitutively transcribes non coding RNA (tRNA and snRNA)
POL I
makes pre rRNAs for the ribosome
what is transcription
making RNA from the DNA instructions: initiation, elongation of RNA chain and termination, pre-mRNA requires processing to become mature mRNA.
Where does transcription start?
+1 site downstream of promoter, polymerisation in 5’-3’ direction
- RNA nucleotides added to 3’ end and reading DNA template strand in 3’ to 5’ direction.
Watson-crick base pairing but replace thymine and uracil. Gene on either strand can overlap (HIV genome)
what direction does polymerisation occur
5’ to 3’ direction, RNA nucleotides are added to the 3’ end and reading DNA template strand in 3’ to 5’ direction.
promoter region tells the cell
Promoter region tells the cell to assemble all the things that are needed for transcription but doesnt actually start transcribing
Where does transcription start? Initiator:
short consensus sequence 2-7 bp long, found at transcription start site of DNA
transcription start site
the precise location on a DNA molecule where RNA polymerase initiates the process of transcription. +1 position ADENINE is where transcript starts, consensus in mammals.
Core promoters
short consensus sequence 6-10 bp found in highly transcribed genes (DNA) sites 26-31 nt upstream of transcription start and defines template strand.
Where is the spot where the protein complexes assemble to start transcription
promoter -> short consensus sequence (6-10bp) highly transcribed genes. 26-31 nt upstream
how many nucleotides do core promoters sit upstream of transcription start
26-31 nt upstream.
CpG islands
initiate transcription of ~70% of protein coding genes, 100-1000 bp long (overlap transcription start and translation start further from promoter region), Very rich in C and G compared to gene body. C METHYLATION SUPPRESSES TRANSCRIPTION.
what part of the promoter has cytosine methylation and suppresses transcription
CpG islands, way of regulating gene expression, methylation (of cytosine) is used as a way of regulating gene expression (can go wrong remember in deamination), stops interaction of the transcription transcriptional proteins with this area and turns gene exp off
What proteins perform transcription
THE PIC: pre initiation complex.
THE PIC pre intiation complex CONSISTS OF
RNA polymerase II and general transcription factors (TFIIE and TFIIH): helicase activity and DNA melting (closed to open)
where does the pre initiation complex assemble and what does it interact with.
Assembles at the promoters (promoter region and interacts with enhancers (DNA loop).
why the pre initiation complex? why general transcription factors?
POLY II is doing the polymerase → adding RNA nucleotides on but cant do anything else so requires general transcription factors.
RNA POLYMERASE II (Structure)
performs polymerisation, adds RNA nulceotides to 3’ end of RNA, reads DNA temp strand in 3’-5’ direction. Unique alpha subunits compared to pol I and III (recognise general transcription factors). Beta subunit clamp domain (c-terminal domain CTD) 7 aas (serine rich) repeated in tandem, serine phosphorlyation is main checkpoint for transcription
beta subunit clamp domain
c-terminal domain CTD: 7 aas (serine-rich) repeated in tandem: serine phosphorylation is the main checkpoint for transcription * point at which all regulation occurs for RNA polymerase
How does RNA polymerase II performs polymersation
adds RNA nucleotides to 3’ end of RNA and reads DNA template strand in 3’ to 5’ direction.
Alpha subunits RNA polymerase II compared to I and III
Polymerase II has unique alpha subunits compared to pol I and III
transcription factors
like co-pilots of RNA pol, all neccessary non polymerase functions, tell RNA Pol II where and when to express specific genes, general tf is TFII
what do transcription factors do
tell RNA polymerase II where and when to express specific genes, all necessary non polymerase functions (where template strand, promoter, start transcription and speed).
what is the key regulatory step of transcription
initiation
Transcription initation
RNA POL II must find correct gene, time and location, low processivity enzyme as it CTD hasn’t been phosphorylated. Maincheckpoint, assembly of TFIIA,B RNA POL II, TFIIF, E and H.
TFIIE
in initiation, binds to the protein complex and acts a docking site for TFIIH
TFIIH and promoter clearance
TFIIH helicase/protein kinase sits ahead of Pol II, melts/unwinds DNA then phosphorylates the CTD, major conformational change in DNA CLAMP -> allows promoter clearance -> release all transcriptional initators & allow high processivity -> fast (50-80 nts per sec) genes transcribed in seconds/mins
what clears the promoter (promoter clearance)
TFIIH helicase/protein kinase sits ahead of pol II melts/unwinds DNA then phosphorylates the CTD -> major confromational change in DNA clamp allowing promoter clearance
once the promoter clearance occurs is transcription fast or slow?
fast, release of all transcriptional initators allows high processivity, RNA pol will transcribe fast (50-80 nts per sec) genes are transcribed in seconds to mins.
transcription elongation
CTD phosphorylation and conformational change, tightly bound substrate (DNA), high processivity enzyme rapidly elongates new pre-mRNAs (further processing needed before mRNAs translated)
stages of initation of transcription
- recognising and binding to the promoter,
- melt DNA
- limited polymerisation
transcription steps
- initation
- elongation
- termination
termination
termination coupled to mRNA cleavage and polyadenylation (poly(A) site and termination sites).
Order of proteins assembling in initation phase
OTHER TFIIs first (A,B), then RNA POL II binds, TFIIE binds to the C-terminal DOMAIN acting as the docking site for TFIIH also on the C-terminal domain.
mRNA processing order
5’ capping
cleavage at the PolyA site
polyadenylation
Co transcriptional mRNA processing
while the mRNA is being transcripted the mRNA is being modified by the enzymes hosted by the CTD (C-terminus domain)
C-terminus domain enzymes
it is long, so not only does it undergo phosphorylation, it also can house enzymes that modify the mRNA for 5’ capping, splicing, cleavage and polyadenylation.
5’ capping
fast, 7 methylguanylate cap on the 5’ end for protection against degradation, all RNA from pol II capped. Joins to 5’ triphosphate masks it from exonuclease attack. Important for translational initation
what does the 7-methylguanylate cap do in 5’ capping
it protects the 5’ end of the mRNA from degradation and MASKS and joined to the 5’triphosphate from exonuclease attack.l
cleavage at poly A site
transcription goes past stop codon and where primary transcript ends, 2 poly A signals (one 10-35 nt upstread 5’ AAUAAA and one 20 nt downstream 3’ GU/U) bound by cleavage factors that bind to each other then cleave the mRNA at the polyA site
poly A signal (5’)
10-35 nt upstream with AAUAAA
Poly A signal (3’)
20 nt downstream with GU/U concentrated
what is left after mRNA cleavage at the poly A site
capped mRNA that does not have extra mRNA transcript in it after the cleavage at the poly A site.
polyadenylation polymerase
polyA polymerase, binds to mRNA after cleavage factors
polyadenylation
polyA polymerase binds to mRNA after cleavage factors, adds 100-200 nt polyA tail that prevents degradation, enhances translation and for mRNA nuclear export. SLOW TO FAST.
is polyadenylation slow or fast
it starts out slow and then becomes rapid.
why does the poly a tail vary in size
some mRNAs get paused at this point resulting in a shorter tail..
is the poly A tail protecting degradation against endonucleases or exonucleases
exonucleases as they cut off the ends of mRNA
Termination of transcription
once mRNA is cleaved off, RNA pol II keeps transcription ~2kb off polyA site, RNA fragment is not capped or polyadenylated and is degraded back to pol II. The CTD will be dephosphorylated and enzymes will fall off mRNA.
where does termination of transcription happen after poly A site cleavage?
within ~2kb of polyA site
what controls the RNA spatial conformation in splicing?
proteins for winding and unwinding of RNA to allow splicing
Spliceosome molecular machine
as big as ribosome, U1-U6 no U3. SnRNPs + proteins. RNA as enzyme and recognise intronic splice sites.
what forms the spliceosome
snRNPs, U1, U2, U4, U5 and U6 + other proteins
snRNPs and components
small nuclear ribonucleoproteins, contain small nuclear RNA and proteins
