Quiz 4 Flashcards
1
Q
what part of the oligo is labeled
A
5’ end
2
Q
what is a “probe” in northern blotting
A
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
3
Q
why are nuclei removed in northern blotting
A
to avoid contamination of the pre-mRNA
4
Q
how is cytoplasmic mRNA isolated
A
taking advantage of the polyA tail that is only on mRNA and hybridizing it to an oligo(dT) tract on a column.
5
Q
what is the bulk of cytoplasmic RNA
A
rRNA and tRNA
6
Q
what does northern blotting allow you to do
A
- 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
7
Q
why can northern blot give quantitative information about the level of expression
A
because the amount of probe that will bind is a function of the target molecules
8
Q
what is the goal of PCR
A
make large quantities of a specific piece of DNA
9
Q
where does PCR reaction add nucleotides
A
at the 3’ OH end
10
Q
when do you get a copy of desired DNA sequence in PCR
A
after three reactions
11
Q
temperature set points in PCR
A
Denaturing: 95 degrees C
extension (thermoresistant DNA polymerase extends from 3’): 72 degrees C
Annealing: 50-60 degrees C
12
Q
why do we use PCR
A
detection of carrier for genetic diseases, single nucleotide polymorphisms (SNPs)
13
Q
what is the result of SNPs (single nucleotide polymorphisms)
A
can put you at increased risk for disease but usually won’t actually CAUSE a disease in the way that a mutation would
14
Q
PCR for RNA target method
A
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
15
Q
advantage of real time PCR
A
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.
16
Q
origin firing
A
initiation of DNA replication from a single origin
17
Q
processivity
A
once polymerase binds, doesn’t detach for hundreds of thousands of nucleotides
18
Q
ORC
A
origin recognition complex - it is what attaches to ori and recruits additional proteins that will recruit replication machinery.
19
Q
how does DNA synthesis proceed
A
from a pre-existing primer that provides a 3’ OH where DNA polymerase can add the next nucleotide.
20
Q
DNA primase
A
subunit of DNA polymerase alpha - RNA polymerase that lays down the RNA primer which is then extended by DNA polymerase
21
Q
what is the issue with DNA using an RNA primer
A
can’t have RNA in the DNA – they need to be replaced and then the DNA needs to be ligated.
22
Q
what cleaves off the RNA primer
A
FEN1, a flap endonuclease
23
Q
function of DNA ligase
A
seals “nick” between 5’ end of old Okazaki DNA and 3’ end of “new” okazaki DNA.
24
Q
types of DNA polymerase
A
epsilon: synthesizes leading strand
delta: synthesizes lagging strand
gamma: synthesizes mitochondrial DNA
alpha: synthesis of RNA primer and primer extension to start replication
25
how are DNA polymerases kept on DNA template
protein clamp that gets loaded as soon as replication initiates.
26
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.
27
telomere
end of chromosome - TTAGGG sequence over and over
28
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
29
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.
30
shelterin
telomere repeat sequences bound by telomere specific proteins
31
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
32
telomerase
reverse transcriptase which carries its own RNA template that can extend the lagging strand.
33
do normal undifferentiated somatic cells have telomerase activity
no - therefore they can only undergo a limited number of cell divisions.
34
hayflick limit
normal undifferentiated somatic cells do not have telomerase activity and can only undergo a limited number of cell divisions
35
telomerase and cancer
reactivation of telomerase unchecked so cells will continue to divide unchecked.
36
damage to cells caused by
1. intracellular rxns of hydrolysis
2. methylation
3. reaction oxygen species (ROS)
4. skin cells via UV light
37
exonuclease
3' to 5' that cleaves out DNA
38
intrinsic exonuclease
part of DNA polymerase that will cleave out a mismatched DNA pair before continuing synthesis
39
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.
40
proteins in mismatch repair system
MLH and MSH
41
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
42
what happens if a base is modified
modified bases can pair with the wrong base
43
what repairs modified bases
base excision repair
44
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.
45
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
46
what does UV cause
pyrimidine dimers - pyrimidine rings covalently link to each other causing a kink in DNA which blocks replication and txn
47
what deals with pyrimidine dimers
nucleotide excision repair system or translesion synthesis
48
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
49
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.
50
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.
51
what causes double stranded DNA breaks
exposure to x-rays
52
how are double stranded breaks usually repaired
non homologous end joining (NHEJ) or homologous recombination
53
explain NHEJ
involves imprecise ligation of broken ends
54
explain homologous recombination
the other chromosome copy is used as the basis of repair for the broken one
55
what is BRCA2 involved in
encodes a protein used in repair via homologous recombination
56
what proteins are involved in NHEJ
Ku and Kinases
57
how does homologous recombination work
RAD proteins allow single stranded DNA chromosome and repair.
58
when does homologous recombination work
in S phase and G2 because need another copy so cell needs to be in active division
59
what causes hemophelia A
recombination event leading to inversion of a chromosomal region
60
what catalyzes transposition
transposase enyzme
61
what encodes the transposase enzyme
a gene located on the transposon itself
62
types of transposons
simple: nothing but transposase coding sequence
complex: has some other gene (ex: antibiotic resistance)
63
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.
64
where is the coding sequence for reverse transcriptase in terms of eukaryotic transposons
the retransposon sequence
65
LINES
long interspersed elements - 500,000 of them, termed L1. full thing is 6000 bp long but usually truncated or mutated and non functional.
66
what is the function of L1
code for a protein that has reverse transcriptase activity
67
SINES
short interspersed elements
68
what is the main SINE
Alul - 300 bp that is present in 1 million copies. No coding sequence (IN INTRON!!)
69
what does transposition of Alul depend on
L1 element
70
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
71
what are proteins held together by in their natural state
multiple weak hydrophobic interactions - can also have some disulfide bonds
72
why don't proteins self assemble in the cell without any help
over crowding and high temperature
73
what happens instead of folding in a normal cell if no helpers
proteins aggregate instead of folding
74
3 phases of folding
1. burst (0-5 ms): formation of secondary structure and collapse of hydrophobic core
2. 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)
3. 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
75
molecular chaperons
proteins that bind and stabilize otherwise unstable conformer of another protein and facilitate its correct fate in vivo
76
what can molecular chaperones help with
1. folding
2. oligomeric assembly
3. transport to a particular subcellular compartment
4. controlled switching between active and inactive conformations
77
how do molecular chaperones bind and release proteins
dependent on ATP binding, hydrolysis and nucleotide exchange.
78
are molecular chaperones enzymes
NO! bind weakly to hydrophobic AA - they increase the yield but not the rate.
79
structure of chaperones
7 small subunit lid, two 7 subunit barrels stacked on eachother
80
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.
81
what is chaperone gene transcription controlled by
Hsf which responds to presence of unfolded protein or heat shock or other types of proteotoxic stress
82
what is protein degradation in the cytosol and nucleus mainly accomplished by
proteasome: a large, gated protease
83
how to proteasome substrates get to proteasome
covalent linkage to multiple copies of ubiquitin
84
proteasome structure
central catalytic core and regulatory cap - acces to core via tunnel formed at the ends of alpha subunit rings
85
what enters proteasome
single unfolded polypeptide - degraded processively
86
three activities of eukaryotic proteasomes
1. cleaves after hydrophobic AA (like chymotrypsin)
2. cleaves after basic AA (trypsin)
3. cleaves after acidic AA (peptidyl-glutamyl peptide hydrolyzing activity)
87
what are the two add'l activities of mammalian proteasomes
1. cleave after branched AA
| 2. cleave between neutral AA
88
what does the regulatory complex of proteasames recognize
ubiquitinylated substrates
89
ubiquitin
protein that becomes covalently linked to polypeptides that are substrates for degradation
90
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.
91
what does attachment of ubiquitin require
action of E1, E2, and E3
92
function of E1
carries out ATP dependent activation of C terminal glycine in two step rxn
1. ubiquitin adenylate formed (dissociated anion)
2. transfer of activated ubiquitin to thiol site in E1 (S-H bond)
93
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
94
function of E3
ubiquitin protein ligase - specifies substrate selection
95
how are misfolded proteins identified
molecular chaperones and then targeted to specific ubiquitin ligases.
96
CHIP
C-terminal Hsp Interacting Protein: binds directly to Hsp70 and catalyzes ubiquitinylation of misfolded proteins
97
aggresome
aggregated proteins that are meeting point for molecular chaperones and proteasomes
98
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
99
what are aggregates bound together by
hydrophobic interactions or ordered assemblies of amyloid fibres
100
what clears aggregates
autophagic system as they are inaccessible to proteasome
101
amyloid
conformation of proteins that involves a stacked beta sheet - form fibres that are very stable
102
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
103
tau
microtubule binding protein -- can form aggregates in alzheimer's -- there are hyperphosphorylated
104
parkinson's disease
loss of dopaminergic neurons in substantia nigra - aggregates enriched in alpha-synuclein (lewy bodies)
105
polyglutamine repeat disease
over 36 glutamine (Q) residues - can cause huntington's disease among others
106
function of smooth ER
synthesis of lipids, cytochrome P450 to detox in liver
107
function of rough ER
many ribosomes
108
chemical environment inside vs outside cell
extracellular is oxidizing, intracellular (cytosol) is reducing
109
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
110
what does the signal peptide bind to
signal recognition particle (SRP), attaches while protein is still being translated
111
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.
112
transolocon
aqueous channel where SRP and protein complex binds. Now TLN can happen into ER itself
113
how does translocon open
into membrane itself (sideways) for membrane proteins or goes through membrane vertically
114
what do proteins that require disulphide bonds req
disulphide isomerase (these bonds are frequently in proteins outside the cell)
115
prolyl isomerase
helps folding
116
what is added to proteins entering the ER
glycosylated on aspargine (N linked) via 14 residue carb with mannose, glucose, and N-acetyl-gucosamine.
117
where is the glycosylated complex from
dolichol anchor to substrate protein
118
calnexin
binds to glucose residues on protein in ER until it folds, then it dissociates
119
what is glycosylation important for
1. protein folding
2. protein stability
3. outside of cell can act as recognition complex
120
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
121
phases of the quality control pathway
1. activation of signaling pathway called unfolded protein response (UPR)
2. ER associated degradation (ERAD)
122
UPR
expression of genes that encode ER specific molecular chaperones and components of the ubiquitin/proteasome pathway so they can be destroyed.
123
ERAD
luminal and membrane proteins are retrotranslocated from the ER to the cytosol for degradation by the proteasome. (Can't happen in the ER!).
124
when can ERAD have expression increased
during UPR
125
Golgi structure
flat membranous discs stacked with curved appearance
126
what happens to proteins once inside the golgi
modified by post tln modification
127
two ways of moving in golgi
1. vesicular transport: sequentially in vesicles that go to each stack
2. cisternal maturation - stacks constantly moving. enzymes are what move back
128
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
129
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
130
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
131
pro region
on peptide hormones, trimmed before enzyme becomes active.
132
why do lysosomes look big in lysosomal storage diseases
distended because missing enzymes, not because theyre storing extra stuff
133
what is iron bound to when it comes into the cell
transferrin
134
what is cholesterol endocytosed via
LDL receptor
135
structure of LDL
vesicle contains cholesterol, has phospholipid/cholesterol coat, and ApoB100 protein that is what interacts with the receptor
136
function of clathrin, COPI, COPII
clathrin: TGN to PM or endosomes/lysosomes
COPII: ER to golgi
COPI: golgi back to ER in retrieval pathway
137
what pinches clathrin vesicles off and how can action be inhibited
dynamin - can be inhibited by inhibiting ATPase
138
what actually happens to the clathrin coats that make them bud out
polymerize
139
what does clathrin bind to
adaptin (AP1 or AP2) in the cytosol
140
why must the coat disassemble after being pinched off
THIS is how the targeting happens
141
how do proteins attach to target membrane
RAB proteins attach to tethering proteins
142
RABs
small GTP binding proteins that bind to vesicles and perform a proof-reading function by interacting with specific tethering proteins on target membrane
143
SNARE proteins
target but mostly fuse membranes together
144
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.
145
how do SNARE proteins disassemble
fusion with a specific chaperone, NSF. Needs to be specific because SNARE energy of interaction is so low.
146
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
147
permeability of inner membrane in mitochondria
impermeable
148
what is the cristae rich in
enzymes that form ATP from ADP
149
what is in the intermembrane space
enzymes that phosphorylate other nucleotides apart from ADP
150
function of the matrix
mainly oxidative - contains the mitochondrial genome, ribosomes, tRNAs, and molecular chaperones for folding
151
what does the mitochondrial genome code for
subunits of components of respiratory chain
152
how are mitochondrial proteins targeted
1. 15-35 residue N terminal with basic AA that is cleaved in the matrix by an endoprotease
2. non cleaved internal sequence
153
how is TIM opened
the positive charge on the presequence and use of membrane potential.
154
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
155
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.
156
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 (
157
what is hydrogen peroxide metabolized by in peroxisome
catalase - makes it into water and oxygen
158
what are peroxisomal proteins encoded by
nuclear genes - imported post TLN in FOLDED state into organelle (different than mitochondria which is unfolded)
159
zellwegers syndrome
no import of peroxisomal enzyme
160
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
161
what is the function of RNA pol II
transcribes ribosomal protein genes
162
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!!!
163
NES
type of localization sequence - exit signal, there are also import signals.
164
mechanism of nuclear transport depends on
karyopharins
165
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.
166
GAP
GTPase activating protein - cleaves GTP to GDP
167
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.
168
IF proteins
1. keratins
2. vimentins/desmin
3. neurofilaments
4. nuclear lamins
169
what causes ALS
abnormal accumulation of neurofilaments in axon and cell body or motor neurons
170
what kind of IF can disassemble
nuclear lamins - usually can't disassemble but these get phosphorylated in the cell cycle which allows them to
171
MT
long, hollow tubules - intracellular organization and transport.
172
what do MTs form
mitotic spindle, cilia, flagella.
173
why do MTs have polarity
because dimers prefer to bind to exposed B tubulin surface than alpha tubulin in profilament.
174
where is the + and - end of MTs
+ is beta (this is where it grows!) - is alpha
175
what stabilizes MTs
microtubule associated protein
176
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
177
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
178
dynein vs kinesin
```
kinesin = + end directed
dynein = - end directed
```
179
what is wave action of cilia and flagella propagated by
dynein - gets translated from one to the next
180
how does force generation of actin occur
polymerization and in conjunction with myosin motor proteins
181
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
182
lamellipodia
actin polymerization pushes the cell membrane out and this extension is the lamellipodia
183
filopodia
during movement via actin when it is polymerizing and pushing out
184
four types of cell adhesion molecules
1. cadherins
2. the Ig superfamily
3. integrins
4. selectins
185
what makes up tight junctions
claudins and occludens
186
types of anchoring junction
adherens, desmosome, hemidesmosome
187
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
188
desmosome
bind together on inside of cell and link together with IF
189
hemidesmosome
link together ECM with cell - protein that does linking is integrin. Binds to IF or actin in the case of focal adhesions
190
integrins
cell matrix receptors on cells - abundant on surface and bind ligands with low affinity. alpha and beta subunits held together non covalently.
191
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)
192
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
193
ECM components
GAGs which are usually bound to protein cores to form proteoglycans. also made up of laminin, collagen, fibronectin, and elastin
194
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
195
fibronectin
modular protein that binds to other matrix molecules and receptors on cells. binds to integrin in the cell.
196
anchorage dependence
most cells need to attach for growth and survival - proliferate better when attached and spread out.
197
proteases
break down the matrix which is important for cell migration
198
how are proteases inhibited
1. local activation (need to be activated by other proteases)
2. confinement by cell surface receprots
3. secretion of inhibitors
