Quiz 4 Flashcards
what part of the oligo is labeled
5’ end
what is a “probe” in northern blotting
the 18-25 nt that is complimentary to a sequence on the mRNA in question and gets labeled with a radioactive phosphate. This allows visualization of the mRNA containing the target sequence
why are nuclei removed in northern blotting
to avoid contamination of the pre-mRNA
how is cytoplasmic mRNA isolated
taking advantage of the polyA tail that is only on mRNA and hybridizing it to an oligo(dT) tract on a column.
what is the bulk of cytoplasmic RNA
rRNA and tRNA
what does northern blotting allow you to do
- quantitate transcript levels
- determine whether gene induction is at the transcription level
3 .detect changes in the size of a specific mRNA - detect alternative splicing
why can northern blot give quantitative information about the level of expression
because the amount of probe that will bind is a function of the target molecules
what is the goal of PCR
make large quantities of a specific piece of DNA
where does PCR reaction add nucleotides
at the 3’ OH end
when do you get a copy of desired DNA sequence in PCR
after three reactions
temperature set points in PCR
Denaturing: 95 degrees C
extension (thermoresistant DNA polymerase extends from 3’): 72 degrees C
Annealing: 50-60 degrees C
why do we use PCR
detection of carrier for genetic diseases, single nucleotide polymorphisms (SNPs)
what is the result of SNPs (single nucleotide polymorphisms)
can put you at increased risk for disease but usually won’t actually CAUSE a disease in the way that a mutation would
PCR for RNA target method
use reverse transcriptase (RT) to convert mRNA into cDNA copy and this becomes substrate for PCR. Can use this instead of northern blot in some cases
advantage of real time PCR
include reaction dye like SYBR green which fluoresces when it binds double stranded DNA. Originally amount of input DNA is too low to be detected but will fluoresce as it progresses.
origin firing
initiation of DNA replication from a single origin
processivity
once polymerase binds, doesn’t detach for hundreds of thousands of nucleotides
ORC
origin recognition complex - it is what attaches to ori and recruits additional proteins that will recruit replication machinery.
how does DNA synthesis proceed
from a pre-existing primer that provides a 3’ OH where DNA polymerase can add the next nucleotide.
DNA primase
subunit of DNA polymerase alpha - RNA polymerase that lays down the RNA primer which is then extended by DNA polymerase
what is the issue with DNA using an RNA primer
can’t have RNA in the DNA – they need to be replaced and then the DNA needs to be ligated.
what cleaves off the RNA primer
FEN1, a flap endonuclease
function of DNA ligase
seals “nick” between 5’ end of old Okazaki DNA and 3’ end of “new” okazaki DNA.
types of DNA polymerase
epsilon: synthesizes leading strand
delta: synthesizes lagging strand
gamma: synthesizes mitochondrial DNA
alpha: synthesis of RNA primer and primer extension to start replication
how are DNA polymerases kept on DNA template
protein clamp that gets loaded as soon as replication initiates.
how can viral DNA replication be inhibited
use of selective DNA polymerase inhibitors. EX: AZT in HIV doesn’t have 3’ OH onto which subsequent nucleotide can be added. this is a nucleoside analogue.
telomere
end of chromosome - TTAGGG sequence over and over
T loop
at telomere - DNA folds back onto itself as G rich strand folds back and anneals to C rich strand creating local displacement loop (D loop) resulting in T loop
what is the purpose of T loop
distinguishes telomere from from a broken DNA which would signal cell for apoptosis. This also prevents end to end joining of chromosomes.
shelterin
telomere repeat sequences bound by telomere specific proteins
end replication problem
very end of lagging strand could not be synthesized so would keep getting shorter and there would be an inability to form the T loop
telomerase
reverse transcriptase which carries its own RNA template that can extend the lagging strand.
do normal undifferentiated somatic cells have telomerase activity
no - therefore they can only undergo a limited number of cell divisions.
hayflick limit
normal undifferentiated somatic cells do not have telomerase activity and can only undergo a limited number of cell divisions
telomerase and cancer
reactivation of telomerase unchecked so cells will continue to divide unchecked.
damage to cells caused by
- intracellular rxns of hydrolysis
- methylation
- reaction oxygen species (ROS)
- skin cells via UV light
exonuclease
3’ to 5’ that cleaves out DNA
intrinsic exonuclease
part of DNA polymerase that will cleave out a mismatched DNA pair before continuing synthesis
if the intrinsic nuclease doesn’t catch the error, what will
the mismatch repair system - only degrades the one nucleotide area and adds a new one via DNA polymerase delta.
proteins in mismatch repair system
MLH and MSH
how does MMR system work
recognizes mistake, chews back from 3’ OH end at “nick”,, and then DNA polymerase will syntehsize a new, correct strand
what happens if a base is modified
modified bases can pair with the wrong base
what repairs modified bases
base excision repair
how does base excision repair work
glcolysases that recognize unnatural bases in DNA (ex: uracil glycolase) cut the bond between base and 1’C of ribose sugar to create an abasic site. Abasic nucleotide removed and correct nucleotide is filled in.
what happens to methylated bases in terms of repair
can be repaired by direct reversal wherein a protein binds to the methylated base and transfers the methyl group to a cys residue in its active site
what does UV cause
pyrimidine dimers - pyrimidine rings covalently link to each other causing a kink in DNA which blocks replication and txn
what deals with pyrimidine dimers
nucleotide excision repair system or translesion synthesis
what does the nucleotide repair system deal with and how does it work
pyrimidine dimers. XP protein recognizes distorted DNA region, other XP proteins unwind and excise this patch of DNA, filled in by DNA pol epsilon or delta
what is translesion synthesis
different DNA polymerases that are not processive and are error prone replicate past the pyrimidine dimer, either putting correct or incorrect bases opposite dimer.
why is translesion synthesis OK even though it adds incorrect bases often
disadvantage of an incorrect base is far outweighed by the disastrous effect of a block to replication that would occur if there was no repair and translesion synthesis.
what causes double stranded DNA breaks
exposure to x-rays
how are double stranded breaks usually repaired
non homologous end joining (NHEJ) or homologous recombination
explain NHEJ
involves imprecise ligation of broken ends
explain homologous recombination
the other chromosome copy is used as the basis of repair for the broken one
what is BRCA2 involved in
encodes a protein used in repair via homologous recombination
what proteins are involved in NHEJ
Ku and Kinases
how does homologous recombination work
RAD proteins allow single stranded DNA chromosome and repair.
when does homologous recombination work
in S phase and G2 because need another copy so cell needs to be in active division
what causes hemophelia A
recombination event leading to inversion of a chromosomal region
what catalyzes transposition
transposase enyzme
what encodes the transposase enzyme
a gene located on the transposon itself
types of transposons
simple: nothing but transposase coding sequence
complex: has some other gene (ex: antibiotic resistance)
prokaryotic transposons vs eukaryotic transposons
prokaryotic: DNA - through plasmids etc
eukaryotic: through RNA intermediate - DNA sequence transcribed by RNA polymerase, creating RNA copy. Reverse transcriptase converts this RNA to double stranded DNA which then integrates into target DNA via integrase protein.
where is the coding sequence for reverse transcriptase in terms of eukaryotic transposons
the retransposon sequence
LINES
long interspersed elements - 500,000 of them, termed L1. full thing is 6000 bp long but usually truncated or mutated and non functional.
what is the function of L1
code for a protein that has reverse transcriptase activity
SINES
short interspersed elements
what is the main SINE
Alul - 300 bp that is present in 1 million copies. No coding sequence (IN INTRON!!)
what does transposition of Alul depend on
L1 element
VNTR
variable number of tandem repeats - repeated sequences in contiguous copies. different individuals have different number of repeats. can use PCR analysis and this to do forensic ID
what are proteins held together by in their natural state
multiple weak hydrophobic interactions - can also have some disulfide bonds
why don’t proteins self assemble in the cell without any help
over crowding and high temperature
what happens instead of folding in a normal cell if no helpers
proteins aggregate instead of folding
3 phases of folding
- burst (0-5 ms): formation of secondary structure and collapse of hydrophobic core
- intermediate phase (5-100 ms): involves formation of molten globule intermediate, which has characteristics of both folded and unfolded proteins. (secondary structures finding each other)
- Protein folding and attainment of native structure (rate limiting): conversion of the molten globule via global repacking of hydrophobic side chains and association of domains that were folded independently in intermediate phase
molecular chaperons
proteins that bind and stabilize otherwise unstable conformer of another protein and facilitate its correct fate in vivo
what can molecular chaperones help with
- folding
- oligomeric assembly
- transport to a particular subcellular compartment
- controlled switching between active and inactive conformations
how do molecular chaperones bind and release proteins
dependent on ATP binding, hydrolysis and nucleotide exchange.
are molecular chaperones enzymes
NO! bind weakly to hydrophobic AA - they increase the yield but not the rate.
structure of chaperones
7 small subunit lid, two 7 subunit barrels stacked on eachother
how do proteins get inside chaperonin
unfolded proteins bind to rim of barrel and are displaced into cavity by the lid structure. Protein can then fold in sequestered environment of the chamber. Lid dissociates due to changes in conformation of large subunit as ATP is hydrolyzed.
what is chaperone gene transcription controlled by
Hsf which responds to presence of unfolded protein or heat shock or other types of proteotoxic stress
what is protein degradation in the cytosol and nucleus mainly accomplished by
proteasome: a large, gated protease
how to proteasome substrates get to proteasome
covalent linkage to multiple copies of ubiquitin
proteasome structure
central catalytic core and regulatory cap - acces to core via tunnel formed at the ends of alpha subunit rings
what enters proteasome
single unfolded polypeptide - degraded processively
three activities of eukaryotic proteasomes
- cleaves after hydrophobic AA (like chymotrypsin)
- cleaves after basic AA (trypsin)
- cleaves after acidic AA (peptidyl-glutamyl peptide hydrolyzing activity)
what are the two add’l activities of mammalian proteasomes
- cleave after branched AA
2. cleave between neutral AA
what does the regulatory complex of proteasames recognize
ubiquitinylated substrates
ubiquitin
protein that becomes covalently linked to polypeptides that are substrates for degradation
where does ubiquitin link
in linear chains where the carboxyl end of the terminal glycine becomes covalently attached to epsilon amino group of lysine 48.
what does attachment of ubiquitin require
action of E1, E2, and E3
function of E1
carries out ATP dependent activation of C terminal glycine in two step rxn
- ubiquitin adenylate formed (dissociated anion)
- transfer of activated ubiquitin to thiol site in E1 (S-H bond)
function of E2
ubiquitin conjugating enzyme - accept ubiquitin from E1 and transfer it to the protein substrate in a reaction that requires E3
*essentially catalyzes reaction of transferring UB to substrate
function of E3
ubiquitin protein ligase - specifies substrate selection
how are misfolded proteins identified
molecular chaperones and then targeted to specific ubiquitin ligases.
CHIP
C-terminal Hsp Interacting Protein: binds directly to Hsp70 and catalyzes ubiquitinylation of misfolded proteins
aggresome
aggregated proteins that are meeting point for molecular chaperones and proteasomes
when do aggregates occur and why
when misfolded proteins overwhelm the ubiquitin/proteasome pathyway - keep them together and secluded so that they don’t damage the cell
what are aggregates bound together by
hydrophobic interactions or ordered assemblies of amyloid fibres
what clears aggregates
autophagic system as they are inaccessible to proteasome
amyloid
conformation of proteins that involves a stacked beta sheet - form fibres that are very stable
alzheimers disease
extracellular amyloids of a alpha-beta peptide cleaved from alzheimer’s precursor protein.
intracellular deposits or neurofibrillary tangles, of the microtubule binding protein, tau that is hyperphosphorylated
tau
microtubule binding protein – can form aggregates in alzheimer’s – there are hyperphosphorylated
parkinson’s disease
loss of dopaminergic neurons in substantia nigra - aggregates enriched in alpha-synuclein (lewy bodies)
polyglutamine repeat disease
over 36 glutamine (Q) residues - can cause huntington’s disease among others
function of smooth ER
synthesis of lipids, cytochrome P450 to detox in liver
function of rough ER
many ribosomes
chemical environment inside vs outside cell
extracellular is oxidizing, intracellular (cytosol) is reducing
how are newly made proteins targeted to the ER membrane
N terminal signal peptide - enriched in hydrophobic AA and is often cleaved after import into ER though sometimes there is internal targeting sequence that doesn’t get cleaved
what does the signal peptide bind to
signal recognition particle (SRP), attaches while protein is still being translated
how does SRP works
samples newly made proteins, finds right one, attaches while still being translated and arrests translation. Binds to ER via an SRP receptor complex which is adjacent to translocon.
transolocon
aqueous channel where SRP and protein complex binds. Now TLN can happen into ER itself
how does translocon open
into membrane itself (sideways) for membrane proteins or goes through membrane vertically
what do proteins that require disulphide bonds req
disulphide isomerase (these bonds are frequently in proteins outside the cell)
prolyl isomerase
helps folding
what is added to proteins entering the ER
glycosylated on aspargine (N linked) via 14 residue carb with mannose, glucose, and N-acetyl-gucosamine.
where is the glycosylated complex from
dolichol anchor to substrate protein
calnexin
binds to glucose residues on protein in ER until it folds, then it dissociates
what is glycosylation important for
- protein folding
- protein stability
- outside of cell can act as recognition complex
Type I and Type II membrane proteins
1: N terminus in lumen of ER
2. C terminus in lumen of ER
OR topologically complex with membrane spanning regions
phases of the quality control pathway
- activation of signaling pathway called unfolded protein response (UPR)
- ER associated degradation (ERAD)
UPR
expression of genes that encode ER specific molecular chaperones and components of the ubiquitin/proteasome pathway so they can be destroyed.
ERAD
luminal and membrane proteins are retrotranslocated from the ER to the cytosol for degradation by the proteasome. (Can’t happen in the ER!).
when can ERAD have expression increased
during UPR
Golgi structure
flat membranous discs stacked with curved appearance
what happens to proteins once inside the golgi
modified by post tln modification
two ways of moving in golgi
- vesicular transport: sequentially in vesicles that go to each stack
- cisternal maturation - stacks constantly moving. enzymes are what move back
how do ER proteins mistakenly in golgi get back
receptor recognizes special peptide sequence (KDEL) at C terminus of ER proteins that escaped to golgi - returned to ER after interacting with receptor
how are lysosomal enzymes delivered to lysosome
by secretory pathway - identified in cis golgi by an enzyme that phosphorylates a specific mannose residue of the core carb that was added in the ER (mannose -6-phosphate) Recognized in trans golgi by M6P receptor that sequesters enzymes into specific vesicle for transport to lysosome
importance of pH in lysosome
M6P binding at pH 6 in TGN. Removal of phosphate makes pH 5 - end up with two vesicles, one with receptors and one with cargo that gets delivered to lysosome which has a pH of 5
pro region
on peptide hormones, trimmed before enzyme becomes active.
why do lysosomes look big in lysosomal storage diseases
distended because missing enzymes, not because theyre storing extra stuff
what is iron bound to when it comes into the cell
transferrin
what is cholesterol endocytosed via
LDL receptor
structure of LDL
vesicle contains cholesterol, has phospholipid/cholesterol coat, and ApoB100 protein that is what interacts with the receptor
function of clathrin, COPI, COPII
clathrin: TGN to PM or endosomes/lysosomes
COPII: ER to golgi
COPI: golgi back to ER in retrieval pathway
what pinches clathrin vesicles off and how can action be inhibited
dynamin - can be inhibited by inhibiting ATPase
what actually happens to the clathrin coats that make them bud out
polymerize
what does clathrin bind to
adaptin (AP1 or AP2) in the cytosol
why must the coat disassemble after being pinched off
THIS is how the targeting happens
how do proteins attach to target membrane
RAB proteins attach to tethering proteins
RABs
small GTP binding proteins that bind to vesicles and perform a proof-reading function by interacting with specific tethering proteins on target membrane
SNARE proteins
target but mostly fuse membranes together
how do SNARE proteins work
v-snare (vesicle snare) interacts with t-snare (target membrane snare). These come together and wind and help overcome energy barrier of hydrophilic head groups + water with hydrophobic hydrocarbons. Squeeze out water which decreases energy of interaction.
how do SNARE proteins disassemble
fusion with a specific chaperone, NSF. Needs to be specific because SNARE energy of interaction is so low.
how do proteins get moved in and out of the mitochondria
TOM proteins bring them in the outer membrane, TIM proteins are on the inner membrane and bring it in
permeability of inner membrane in mitochondria
impermeable
what is the cristae rich in
enzymes that form ATP from ADP
what is in the intermembrane space
enzymes that phosphorylate other nucleotides apart from ADP
function of the matrix
mainly oxidative - contains the mitochondrial genome, ribosomes, tRNAs, and molecular chaperones for folding
what does the mitochondrial genome code for
subunits of components of respiratory chain
how are mitochondrial proteins targeted
- 15-35 residue N terminal with basic AA that is cleaved in the matrix by an endoprotease
- non cleaved internal sequence
how is TIM opened
the positive charge on the presequence and use of membrane potential.
what is function of Hsp70 in mitochondrial import
binds the protein when it comes through TIM and pulls it into the matrix - helps it to fold
LHON
leber’s hereditary optic neuropathy - mitochondrial missense mutation in subunit 4 of NADH-coQ reductase. Present at midlife with sudden blindness. SAME mutation can lead to dystonia which is generalized motor disorder.
peroxisome
small single membrane organelles that get their name from metabolism of hydrogen peroxide of organalle. important for fatty acid beta oxidation and plasmalogen synthesis (
what is hydrogen peroxide metabolized by in peroxisome
catalase - makes it into water and oxygen
what are peroxisomal proteins encoded by
nuclear genes - imported post TLN in FOLDED state into organelle (different than mitochondria which is unfolded)
zellwegers syndrome
no import of peroxisomal enzyme
ALD
oxidation of long chain fatty acid is defective - this gene is a membrane transporter for long chain fatty acyl coA synthase from cytosol to peroxisome matrix
what is the function of RNA pol II
transcribes ribosomal protein genes
NLS
nuclear localization sequence - can exist anywhere on the protein but must be accessible to receptors. 4-8 AA rich in arg and lys. DONT GET CLEAVED!!!
NES
type of localization sequence - exit signal, there are also import signals.
mechanism of nuclear transport depends on
karyopharins
karyopherins
recognize NLS or NES sequences. Karyopherin binds RanGTP when it enters the nucleus and unloads cargo, this gets cleaved to GTP to GDP when it exits alone with receptor.
GAP
GTPase activating protein - cleaves GTP to GDP
structure of IF
elongated and alpha helical with globular N terminus and globular C terminal tail. Forms a dimer that is a coiled coil, associate to form a staggered tetramer. 8 tetramers assemble to make filament.
IF proteins
- keratins
- vimentins/desmin
- neurofilaments
- nuclear lamins
what causes ALS
abnormal accumulation of neurofilaments in axon and cell body or motor neurons
what kind of IF can disassemble
nuclear lamins - usually can’t disassemble but these get phosphorylated in the cell cycle which allows them to
MT
long, hollow tubules - intracellular organization and transport.
what do MTs form
mitotic spindle, cilia, flagella.
why do MTs have polarity
because dimers prefer to bind to exposed B tubulin surface than alpha tubulin in profilament.
where is the + and - end of MTs
+ is beta (this is where it grows!) - is alpha
what stabilizes MTs
microtubule associated protein
when does B tubulin hydrolyze bound GTP
when tubulin dimer binds to an MT - when there are plentiful free dimers, hydrolysis occurs after further tubulin is added
how do MTs pull chromosomes apart
dynein like motor proteins on cell membrane, kinesin binds to overlap and pulls, also get dynamic instability at kinetochore
dynein vs kinesin
kinesin = + end directed dynein = - end directed
what is wave action of cilia and flagella propagated by
dynein - gets translated from one to the next
how does force generation of actin occur
polymerization and in conjunction with myosin motor proteins
treadmilling
in regular cells, actin isn’t fixed and the rate of addition at one end is the same as the rate of degradation at the other
lamellipodia
actin polymerization pushes the cell membrane out and this extension is the lamellipodia
filopodia
during movement via actin when it is polymerizing and pushing out
four types of cell adhesion molecules
- cadherins
- the Ig superfamily
- integrins
- selectins
what makes up tight junctions
claudins and occludens
types of anchoring junction
adherens, desmosome, hemidesmosome
adherens junctions
has to do with homophilic interactions between cadherins. binds to receptor inside cell that then binds to actin. makes big connections of actin via alpha and beta catenin
desmosome
bind together on inside of cell and link together with IF
hemidesmosome
link together ECM with cell - protein that does linking is integrin. Binds to IF or actin in the case of focal adhesions
integrins
cell matrix receptors on cells - abundant on surface and bind ligands with low affinity. alpha and beta subunits held together non covalently.
selectins
lectins (carb binding) that mediate calcium dependent cell cell adhesion in the bloodstream. during inflammation, endothelial cells express E selectin that binds to the carbs on white blood cells and platelets (which have L and P selectins)
BL
basal lamina - prevents passing of macromolecules from blood to urine. Prevents fibroblasts from contacting epithelial cells. Does NOT stop macrophages, lymphocytes, and nerve processes from passing through it
ECM components
GAGs which are usually bound to protein cores to form proteoglycans. also made up of laminin, collagen, fibronectin, and elastin
Marfans syndrome mutation
in fibrilin which is part of elastin. usually bound to TGF beta in inactive form but with mutation it isnt so TGF beta is overactive. a lot of growth factor causes the disease
fibronectin
modular protein that binds to other matrix molecules and receptors on cells. binds to integrin in the cell.
anchorage dependence
most cells need to attach for growth and survival - proliferate better when attached and spread out.
proteases
break down the matrix which is important for cell migration
how are proteases inhibited
- local activation (need to be activated by other proteases)
- confinement by cell surface receprots
- secretion of inhibitors
