Exam 5 Flashcards
What is the accepted proposed model of translation?
Adaptor Hypothesis (tRNA adaptors)
What 2 ways is the proper reading frame set?
- start codons
- ribosomes ensure the reading frame is maintained
How many possible codons are there?
64
What are the stop codons?
UAA
UAG
UGA
What is the start codon?
AUG
Multiple codons encode the _______ amino acid (synonyms)
same
_______ molecules serve as adaptors during translation
tRNA
Codon-anticodon pairing is ____________
anti-parallel
What position in the codon is NOT Watson-Crick pairing?
3
What benefit does the third wobble space in a codon have?
more base pairing options
What is responsible for stabilizing all tRNA tertiary structures?
nine long-range base pairs
_________ nucleotides are common in tRNA
unusual
What is amino acid activation (charging)?
attaching amino acid to tRNA
Does tRNA charging require ATP of GTP?
ATP
What are the 2 adaptors in translation?
tRNA synthetase
anticodon of tRNA
What does tRNA synthetase do?
attaches correct amino acid to each tRNA molecule
How is the accuracy of AA-tRNA synthetase maintained?
hydrolytic editing
How does hydrolytic editing work for AA-tRNA synthetase?
The incorrect amino acid is moved up to the editing site where it is proofread and removed
How does AA-tRNA synthetase recognize the correct tRNA?
unique identity elements on the anticodon region, 5’ and 3’ end
Translation in prokaryotes initiates with ____________ and ________
N-Formylmethionine
tRNA-fMET
How do N-Formylmethionine and tRNA-fMET initiate translation?
N-Formylmethionine is added to tRNA-fMET then cleaved away
What adds Met to tRNA(fMET)?
methionyl-tRNA synthetase
What adds Met-tRNA(fMET) to N-formyl?
methionyl-tRNA formyltransferase
What is the prokaryotic ribosome?
50S + 30S = 70S
What is the eukaryotic ribosome?
60S + 40S = 80S
Are prokaryotic ribosome made mainly of protein or rRNA?
rRNA
_____-protein facilitate ribosome function by transmitting information between different rRNA structures
R-proteins
What are the 3 binding sites of ribosomes?
A
P
E
The ____ and ____ site of a ribosome is base paired with their codons while the ____ site is not
A and P
E
What prokaryotic ribosome subunit is Peptidyl Transferase Center located?
50S (large)
Does the Peptidyl Transferase Center use proteins or rRNA to carry out its function?
rRNA
What is the job of the Peptidyl Transferase Center?
mediates peptide bond formation as the chain grows
What end of the growing peptide chain does new residues get added to?
C terminus
What is the job of the IF-1 in prokaryotes?
aids IF-3 in binding and blocks the A site
What is the job of the IF-2 in prokaryotes?
binds fMET-tRNA and GTP to 30S
What is the job of the IF-3 in prokaryotes?
prevents 50S binding and releases mRNA and tRNA
What does the 30S initiation complex consist of?
IF-3
IF-1
fMET-tRNA
GTP
What is the job of the EF-Tu in prokaryotes?
binds AA-tRNA and GTP to A site
What is the job of the EF-T in prokaryotes?
displaces GDP from EF-Tu
What is the job of the EF-G in prokaryotes?
promotes translocation through GTP hydrolysis
What prokaryotic elongation factor proofreads?
EF-Tu
What prokaryotic elongation factor does translocation from A to P site?
EF-G
How does the bottom of the A site in prokaryotes proofread?
checks to make sure there is no bulging in the codon-anticodon pairing
What does RF-1 do?
recognizes stop codons UAA and UAG
What does RF-2 do?
recognizes stop codons UAA and UGA
What does RF-3 do?
releases RF-1 and RF-2 via GTP hydrolysis
What does RFF do?
with EF-G induces ribosome dissociation
Do prokaryotic or eukaryotic mRNAs have 5’ or 3’ modifications?
eukaryotic
Do prokaryotic or eukaryotic mRNAs encode multiple different proteins?
prokaryotic
Do prokaryotic or eukaryotic mRNA contain their own independent ribosome binding sites as well and stop and start codons?
prokaryotic
Do prokaryotic or eukaryotic translation initiate independently at each gene?
prokaryotic
Do prokaryotic or eukaryotic translation usually initiate by a 40S subunit being recruited to the 5’ cap?
eukaryotic
Do prokaryotic or eukaryotic have sequences that regulate translation initiation at 5’ UTR?
eukaryotic
Do prokaryotic or eukaryotic have sequences that regulate mRNA stability at 3’ UTR?
eukaryotic
What are eukaryotic translation initiation factors called?
eIF
In eukaryotes translation initiation is tightly controlled by phosphorylation of _____
eIF2
What carries out the phosphorylation of eIF2 in eukaryotes translation initiation?
guanine nucleotide exchange factor (GEF)
What is the process of eIF2 phosphoryaltion in eukaryotes translation initiation regulation?
eIF2+GDP is phosphorylated to GTP which activates eIF2 allows protein synthesis
Multiple ribosome can translate an mRNA simultaneously in a ____________ complex
polysome
How is translation in eukaryotes in a circular configuration?
eIF4G at the 5’ end binds to poly-A at the 3’ end
What is the benefit of translation of eukaryotes in a circular configuration?
used to regulate the age of an mRNA
What is the only part of intracellular protein trafficking that is transmembrane and not vesicular?
cytosol to ER
________ is maintained during vesicular transport
topology
Does the ER have more membrane or more empty space?
more membrane
How many membranes does the ER have?
one
What is the purpose of the smooth ER?
lipid synthesis
What is the purpose of the rough ER?
entry point for proteins into the secretory pathway
What are translational elements?
(apart of the rough and smooth ER) sites where transport vesicles bud off to cary newly synthesized proteins and lipids to the golgi
Disrupted ER membranes form ________
microsomes
What are microsomes?
When ER membrane is disrupted and forms a circular vesicle with its membrane
Where is the SER and RER located in the sucrose concentration when separating them?
rough ER: high density sucrose
smooth ER: low density sucrose
What are the 2 types of ribosomes?
free (protein not translated into ER)
membrane bound
When separating free and membrane bound ribosomes, where are they each located in the sucrose concentration?
free: low density sucrose
bound: high density sucrose
What mRNAs are attached to membrane bound ribosomes?
secreted proteins going to ER
What mRNAs are attached to free ribosomes?
cytosolic proteins (not going to ER)
What are the 4 secretory signals that target protein to the ER?
- positive amino terminal
- alpha helix core
- turn inducing residues (helix breaking)
- specific cleavage site
Basic residues at the N terminus of signal anchors the signal to the cis (outside) face of membrane preventing ____________ causing a insertion loop
headfirst insertion
What is the evidence for proteinaceous channels for protein insertion instead of a lipid lined pore?
If there is a protein channel and you prematurely remove the protein you would be able to see ions flowing through
What is the Original Signal Hypothesis?
protein is fed into the ER with N terminus still on the outside forming a hairpin inside the ER and once all is inside signal peptidase cleaves the peptide
What are the 2 stages of ER translocation?
- targeting of polypeptide to ER surface
- translocation into the ER membrane (requires ATP)
What does SRP do in ER translocation?
recognizes the signal peptide coming out of the ribosome (tip of polypeptide) and brings to the SRP receptor on ER surface
What targets the signal peptide to the ER surface?
SRP
Once about 20 amino acids on the chain emerge from the ribosome bound to SRP what happens before SRP docking?
translational arrest
When is the translational arrest reversed in ER translocation?
When the SRP docks on the STP receptor on the ER membrane
ER translocation in vitro requires _________ since most proteins are inserted co-translationally
microsomes
What would happen to the protein if SRP is present but no SRP receptor?
It wont be made because the SRP doesn’t think its made it to the ER since the receptor isn’t present
What are the 3 subunits of SRP?
p19/54
p68/72
p9/14
What does the p19/54 subunit of SRP do?
binds to signal sequence
What does the p9/14 subunit of SRP do?
binds to the A site of the ribosome and arrest translation
What does the p68/72 subunit of SRP do?
helps initiate insertion of the signal sequence into the ER membrane by binding to SRP receptor
SRP increases in binding affinity when the ____________ is present
signal sequence
What subunit of SRP is a GTP binding domain (GTP hydrolysis)?
p54
_______ receptor is much less abundant that ribosomes on the RER surface suggesting its not involved in the earlier stages
SRP receptor
Is co-translational or post-translational ER translocation more common?
co-translation
What does Sec61p do as an ER translocase?
helps anchor the ribosome to the ER membrane (interacts directly with translocating polypeptide)
Crosslinking of Sec61p to translocating polypeptides requires _______
ATP
What does Bip do as an ER translocase?
binds to misfolded protein and refolds them and crosslinks and pulls polypeptide through
Are Sec62p and Sec63p required from ER translocation?
No
_________ and _______ are sufficient alone for protein translocation into the ER
Sec61p and SRP receptor
What does the signal peptidase do in ER translocation?
cleaves the signal sequence
What does Oligosaccharyltransferase do in ER translocation?
glycosylation
Is signal peptidase or oligosaccharyltransferase required for ER translocation?
No
What 3 factors influence the protein topology during ER translocation?
- proteolytic processing
- location of signal
- charge around the signal
What is Type I topology of integral membrane proteins?
N terminus on trans side (lumen)
What is Type II topology of integral membrane proteins?
N terminus on cis side (cytosol)
What is Type III topology of integral membrane proteins?
two or more membrane spanning segments
Is the cis side located on the lumen or cytosol?
cytosol
Is the trans side located on the lumen or cytosol?
lumen
What 2 ways are different topological orientation obtained?
targeting signals on polypeptide
start and stop transfer sequences (polytopic)
Orientation of proteins across the membrane is mediated by hydrophobic stretches of amino acids called _______________
start and stop transfer sequences
Start and stop transfer sequences are _________ independent (they start with it then don’t need it again)
SRP-independent
Start and stop transfer sequences have clearly defined _________ sections that stay inside the membrane
hydrophobic
Normally, start transfer sequences have _______ residues before them that determine the orientation of the signal insertion (N towards cytoplasm)
basic
In some cases, the direction of insertion of the start sequence can be reversed (N towards lumen) if the start transfer signal is followed by _______ residues
basic
When integrating proteins in the ER membrane where does the negative charges typically point?
lumen
When integrating proteins in the ER membrane where does the positive (basic) charges typically point?
cytosol
Proteins that are not properly folded or modified in the ER are subject to __________
ERAD
What happens during ERAD?
protein is transported retrograde from ER to cytoplasm to be degraded
The ER lumen contains a lot of Bip why?
it binds to hydrophobic regions to make sure proteins are folding correctly
___________ prevents disulfide bond formation in the cytosol and catalyzes them in the ER
Glutathione
Are disulfide bonds preferred in the cytosol or ER?
ER
In the cytosol what is the GSH:GSSG ratio?
50:1 DOES NOT promote disulfide bonds
In the ER what is the GSH:GSSH ratio?
5:1 optimal for disulfide bonds
What is Protein Disulfide Isomerase (PDI)?
make sure improper disulfide bonds are not created and creates correct bonding
What is an example of an soluble protein ER retention signal?
KDEL
What is an example of an transmembrane protein ER retention signal?
KKXX
What is the Unfolded Protein Response (UPR)?
Decreases translation of the misfolded protein and increases gene expression of genes that help fix the unfolded proteins
What are the 2 ER quality control mechanisms?
ERAD
UPR
What are the 3 main players in the UPR in yeast?
Ire1p
HAC1 mRNA
Rlg1p tRNA ligase
During the UPR in yeast, ER stress induced ____ to oligomerize and autophosphorylate which activates it
Ire1
During the UPR in yeast once Ire1p is autophosphorylated and activated, _________ is made
Hac1
What player in the UPR in yeast enters the nucleus and activates transcription of genes needed?
Hac1p
What are the 3 main players in the UPR in mammals?
Ire1p
ATF6
PERK
In the UPR in mammals _______ splices XBP1 then XBP1 can enter nucleus and activate transcription
Ire1p
In the UPR in mammals, _______ is cleaved directly from cytoplasmic domain of ER and contains transcription factors
ATF6
In the UPR in mammals, translational repression is mediated by ____________
PERK
In the UPR in mammals, _______ is analogous to HAC1 in yeast
XBP1
When _____ is bound to IRE1, PERK, and ATF6 in the mammalian UPR the response is off
Bip
During the UPR, _____ usually cannot enter the nucleus because its bound to the ER membrane but when UPR is activated it can enter
ATF6
During regulation of translation in the UPR, ______ phosphorylation inhibits bulk translation
elF2-alpha
What is the molecular issue with cystic fibrosis?
single amino acid is mutated and the ER wont let the protein leave the ER
What 3 places are ribosomes found?
ER
cytosol
mitochondria
The ________ and ________ pathways are connected
secretory and endocytic
Vesicle bud from _______ compartment and fuse with ______ compartment
donor
target
_________ is the space within membrane enclosed compartments
lumen
How does COP II coat proteins transport?
anterograde (ER to cis Golgi)
How does COP I coat proteins transport?
retrograde (Golgi to ER)
How does clatherin coat proteins transport?
between endosomes and golgi from plasma membrane
What energy is required for coat proteins?
GTP
What do inner adaptor proteins bind?
transmembrane cargo receptors (with exit signals)
outer coat proteins
Inner coat proteins form __________ and outer coat proteins help with ________-
vesicles
budding
What are the two GTP binding protein involved in phosphorylation of coat proteins?
GEF and GAP
In coat protein phosphorylation, GEFs activate or inactivate?
activate (protein kinase)
In coat protein phosphorylation, GAPs activate or inactivate?
inactivate (phosphatase)
What is the GTP cascade of GEFs?
release of GDP and binding of GTP
What is the GTP cascade of GAPs?
GTP hydrolysis to GDP
Proteins leave the ER in _______ coated vesicles
COP II
Inner coat adaptor proteins help recruit _____ coat proteins to help bud vesicles
outer
Membrane _______ event pinches off vesicle
fusion
What provides quick movement for vesicles between ER and Golgi?
microtubule networks
COP___ coat dissembles as vesicles transport anterogradely to golgi
COP II
COP ___ coat assembles as vesicles transport retrogradely to ER
COP I
High affinity for KDEL sequence is present in ___________ and __________
vesicular tubular clusters
Golgi
Low affinity for KDEL sequence is present in ________
ER
Why is the KDEL affinity low in the ER?
because ER proteins aren’t suppose to leave the ER anyways so having a lower KDEL affinity means its less likely to leave the ER
Why is the KDEL affinity high in the vesicles and golgi?
these proteins are meant to be secreted and higher affinity means transport
High affinity of KDEL corresponds with COP ___ because…
COP I
retrograde transport
Rab proteins are important for ______ vesicles to target membrane
tethers
SNARE proteins are important for _____ vesicles to target membrane and ______- of opposing lipid bilayers
dock
fusion
What are the 2 types of SNARES?
v and t
______ ensure organelle specific targeting or vesicles not _____
Rabs
SNARES
______ cascade is important for vesicle maturation
Rab
The Rab cascade for vesicle maturation is regulated by ___ and ____
GEFs and GAPs
What are the 3 steps of vesicles fusion using SNAREs?
- ATPase disassembles paired v and t SNAREs
- v and t SNARES from adjacent vesicles interact
- membranes fuse
What part is the entry to the Golgi closest to the ER?
cis
What part of golgi takes vesciles from the ER?
cis
What is the stackable membrane enclosed compartments in Golgi?
cisterna
What is the exit point for proteins in the Golgi?
trans
What starts N-linked glycosylation in ER?
GlcNac
What is the purpose of ER glycosylation?
proper protein folding
removal of misfolded proteins
What starts O linked glycosylation in Golgi?
GalNac
What are the steps of Oligosaccharide processing?
- ER: removal of glucose and mannose
- Golgi: removal of 3 mannose
- Golgi: addition of GlcNac
- Golgi: removal of 2 mannose
- Golgi: addition of sugars
What is cause of Congenital Disorders of Glycosylation (CDG)?
deficiencies in genes related to N-linked glycosylation
T/F? ST6Gal1 is upregulated in certain cancers
true
What is Triskelion?
shape of one clatherin that comes together to form pits
_______ protein bind to membrane bound proteins and the triskelion in clatherin coated vesicles
adaptor
Coat assembly of clatherin induces membrane _________
curvature
__________ proteins are recruited to neck of budding clatherin vesicles
fission
What energy is required for clatherin coating?
GTP
Are all coat protein GTP binding mediated?
yes
During protein sorting in the trans golgi networks, __________ tags proteins for delivery to lysosomes
M6P
What is the difference between regulated and constitutive secretory pathway?
regulated: proteins guided to secretory vesicles in specialized cells
constitutive: common proteins delivered to plasma membrane
Where is P-GlcNAc added to proteins?
cis golgi
Where is GlcNAc removed exposing M6P?
trans golgi
What cleaves GlcNAc exposing M6P on lysosomal hydrolase?
alpha-N-acetylglucosamidinase
What is the most basic in the secretory pathway?
ER
What maintains the low pH in lysosomes?
ATPase H+ pump
What is Neuronal Ceroid Lipofuscinoses (NCL)?
mutations in proteins that compromise lysosome function
What is the most common movement disorder related to lysosome disfunction?
Parkinsons
Is the constitutive or regulated secretory pathway happening all the time?
constitutive
What are the steps of exocytosis of synaptic vesicles?
- vesicles dock to plasma membrane
- t-SNARE assembles
- SNARE bundle assembles blocking complexin protein
- action potential triggers Ca2+ entry
- Fusion block released, fusion pore opens
Is endocytosis clatherin dependent?
yes
What are the 3 possible fates of endocytosed cargo?
- recycled
- transcytosis
- degradation
Endocytosis of clathrin coated vesicles form clathrin coated ________
pits
What are MVB? (multivesicular bodies)
endosomes with multiple vesicles in them
Diabetes affects the ________ of glucose
endocytosis
Is macropinocytosis clathrin dependent or independent?
independent
What is macropinocytosis?
When cell membrane protrudes and swallows up things outside of cell
What is caveolae?
pits formed in cell membrane and are swallowed by cell and pinched off
Is caveolae clatherin dependent or independent?
independent
_________ coated vesicles are responsible for delivering cargo from the TGB to endosomes; from endosomes to TGN; and from the plasma membrane to endosomes
clathrin
The _______ pathway is critical for delivery of hydrolases to lysosomes
M6P
o
MVB (multivesicular bodies)
