Exam 5 Flashcards
(216 cards)
1
Q
tertiary protein structure
A
formation of domains
2
Q
when does protein folding assistance beginning
A
as soon as peptide emerges
3
Q
denturants
A
detergents (SDS), chaotropic slats (NaSCN, NaClO4), high temp, Urea, guanidine HCl
4
Q
Anfinsen’s experiment
A
reduce (BME) then denature (8M Urea) RNaseA (4 S-S). dialysis (remove urea). Oxidize back to S-S. All correct S-S. Swap dialysis and oxidation steps. Random S-S.
5
Q
lead to protein aggregation (3)
A
hydrophobic region exposed. Very high concentration. hydrophobic regions of monomers binding one another.
6
Q
Alzheimers protein folding
A
alphabeta protein Beta sheets adhere and form long fibrils
7
Q
e. coli chaperone
A
DnaK (Hsp70 analog), GroES-GroEL (Hsp 60)
8
Q
eukaryote chaperone
A
Hsp 70, Hsp90
9
Q
Hsp70 function
A
bind hydrophobic regions. uses ATP to prevent misfolding. allows time for productive folding
10
Q
nascent protein 1st met by ? as it leaves ribosome. name pro and euk. Transfers to Hsp70 if needed.
A
TF (pro) , NAC (euk)
11
Q
three protein folding pathways
A
chaperone independent. Hsp70 assisted. Hsp70 + chaperonin
12
Q
Dnak (Hsp70 homolog) regions
A
N t-term ATP binding. C-term peptide binding
13
Q
Hsp70 (DnaK) mechanism
A
DnaJ delivers unfolded or partially folded peptide to DnaK-ATP. Hydrolysis of ATP prevents RAPID folding. GrpE replaces ADP with ATP and partially folded peptide dissociates. Peptide may finish folding independently OR require another cycle OR require GroEL (chaperonin)
14
Q
GroEL structure/function
A
aka Anfinsens cage. hydrophobic inner regions interact with substrate. Recruits GroES cap. 7 ATP binding sites in central and lower GroEL cavity
15
Q
GroEl-GroES mechanism
A
peptide binds hydrophobic region in GroES central cavity. 7 ATP and GroES cap bind GroEL central cavity. ATP hydrolzyed over 15 seconds. 7 more ATP bind lower GroEL cavity. ADP and GroES dissociate. folded peptide dissociates.
16
Q
proteins are delivered to cellular compartments by ?
A
translocation
17
Q
characteristics of translocated prokarotic proteins
A
preproteins with N-terminal leader sequence
18
Q
eukaryote synthesis of secretory and membrane proteins coupled to translocation across ?
A
ER membrane
19
Q
participate in secretory protein translocation
A
SRP (signal recognition particles), SR (signal receptors), and translocons
20
Q
modifies and sorts secretory and membrane proteins
A
golgi
21
Q
golgi function for secretory and membrane proteins
A
modifies and sorts
22
Q
prokaryote N-term leader sequence characteristics
A
total about 26 residues. far N-term (basic residues), central N-term (hydrophobic), C-term of N-term (nonhelical). Also a cleavage site
23
Q
Unique roll of Hsp90
A
functions on regulated “client proteins” to function. (ie. tyrosine kinases, telomerases) Assists binding. uses ATP
24
Q
SecB mediated transloaction
A
SecB binds protein to be translocated. binds SecA/SecYEG on membrane. ATP binds and SecB dissociates. ATP hydrolysis drives protein threading through membrane
25
nuclear peptide targeting sequences
positively charged regions (lysine)
26
ER peptide targeting sequence
hydrophobic region
27
peroxisome targetting sequence
-SKL-
28
secretory protein translocation into ER
signal sequence emerges from ribosome. SRP binds and arrests translation. docks ribosome on ER receptor. GTP dissociates SRP and translation resumes through translocon on ER membrane. signal peptidase in ER lumen cleaves signal sequence. BIP (Hsp70) assists folding. SRP hydrolyzes GTP and is recycled. signal is degraded.
29
fate of ER synthesized proteins
secretion, membrane, lysosome
30
signal recognition particle domains
Alu domain (contains 5' and 3' ends, translation termination), S domain (signal recognition and ER targeting)
31
how does SRP Alu domain cause translation arrest
competes with elongation factors
32
eukaryotic ER translocon components
docking receptor (composed of Sec61 and TRAM), SP (signal peptidase), OST (adds carbohydrate to peptide)
33
major modification by golgi and ER
glycosylation by enzymes on luminal membrane. post golgi localization is often based on glycosylation
34
direction of flow through golgi
cis to trans. post golgi localization is often based on glycosylation
35
CFTR
cystic fibrosis transmembrane conductance regulator. epithelial Cl- channel. Regulates H2O secretion
36
deltaF508 effect on CFTR
prevents proper trafficking to golgi. Does not lose Cl- channel activity. functions normally if forced onto surface
37
mutation of CFTR that prevents proper trafficking to golgi
deltaF508. functions normally if forced onto surface of golgi
38
mitochondrial translocon
multiple translocons depending on destination. All interact with TOM (outer MM translocon). SAM for outer membrane. Tim22 or 23 for inner membrane or matrix respectively.
39
nuclear localization signal targets ?
importin alpha/beta complex
40
nuclear translocation
peotein binds imortin alpha/beta. movement through nuclear pore complex mediated by RanGTPase. cargo dissociates from beta then alpha. beta and alpha shuttled back to cytosol
41
protein modifications that generally occur cotranslationally
glycosylation, acylation
42
modifications that generally occur post-translationally
proteolytic cleavage, phosphorylation, methylation, sulfation
43
most common post translational modification
proteolytic cleavage
44
prenylation
addition of isoprene lipids (unsaturated lipid)
45
purpose of glycosylation
increased solubility. assist folding. localization. extracellular recognition,
46
o-linked glycosylation
glycosylation of OH groups of Serine or threonine
47
where does glyosylation of OH groupos of serine or threonine (o-linked) occur?
exclusively Golgi
48
N-linked glycosylation occurs where?
starts in ER, continues in golgi
49
N-linked glycosylation on ? residues
asparagine
50
o-linked glycosylation target sequence
no target sequence (:
51
n-linked glycosylation target sequence
-NX(S/T)- X is any but proline
52
n-linked pentasaccharide core
two n-acetylglucosamine and three mannose
53
dolichol phosphate function
first recipient of glycosyl residues destined for protein. located in ER membrane. oligosaccharide destined for attachment to Asn
54
enzyme that adds sugar to dolichol phosphate
glycosylaminotransferases
55
blocks initial linkage of sugar to dolichol phosphate. how?
tunicamycin. mimics UDP-GlcNAc. leads to UPR (unfolded protein response)
56
dolichol phosphate glycosylation pathway
glycosyltransferases add two GlcNAc core to cytosolic side of dolichol. Add 5 mannose. Flips oligosaccharide to ER lumen. Further glucose and mannose transferred from lumen dolichol. oligosaccharyl transferase transfers saccharide from dolichol to protein.
57
tunicamycin
mimics UDP-GlcNAc. blocks assembly of pentasaccharide core. lead to UPR (unfolded protein response)
58
unfolded protein response (UPR)
activate PERK kinase. phosphorylates/inhibits eIF2/protein synthesis
59
proteins destined for lysosome are marked with ?
m6P. mannose 6-phosphate
60
marks proteins with m6P (for lysosome localization)
N-acetylglucosamine transferase.. GlcNAc-transferase
61
I cell disease
caused by inclusion bodies from inability to target proteins to lysosome with m6P label
62
prenylation consensus sequence
-CAAX A is aliphatic, X determines prenylation type
63
two types of prenylation and consensus sequence
Ala, Met, Ser (farnesyl), Leu (geranylgeranyl)
64
function of prenylation
anchors to membrane. signal transduction
65
farnesyl linkage is to ?. enzyme? occurs where?
cysteine thiol. farnesyl protein transferase. in cytosol
66
fate of prenyl consensus sequence
proteolysis of AAX in ER. carboxymethylation of C
67
H-ras and N-ras fate after prenylation
also palmitoylated in ER
68
HOT target for treatment of Ras mediated cancers
farnesyl protein transferase
69
myristoylation consensus sequence
amino terminal.. (M)GXXX(S/T)
70
myristoylation general mechanism
occurs cotranslationally. myristic acid activated by CoA attachment. linkage through amide with glycine
71
myristoylated proteins
often found in cytoplasm or membranes. involved in signal transduction
72
palmitoylation consensus
(M)(X_1-9)C. but not really known
73
palmitoylation general mechanism
reversible ester linked acylation through cysteine
74
palmitoylation function
anchors to membrane. signal transduction
75
prokaryotic proteins are degraded by ___ or __
proteasomes or the HtrA protease
76
eukaryotic proteins are degraded by
proteasomes, HtrA protease or lysosomes
77
measure protein half life
pulse chase. pulse 35SMet for various amounts of time. . to other samples, chase with Met. . PAGE autoradiography
78
characteristics of unstable protein in pulse chase
fast 35SMet incorporaton. fast disappearance
79
membrane bound organelle disposal site
lysosome
80
lysosomes contain ? for breakdown. how maintained?
acid hydrolases. uses H+-ATPase pump
81
degrade ~85% of intracellular protein
26S proteasome in euk. ClpAP in prok
82
26S proteasome structure
20S core19S caps select for ubiquitinated proteins (15 different proteins, 6 ATPases)
83
26S proteasome 20S core
4 rings of 7 subunits (2 alpha rings and 2 beta rings). 2 beta rings make up central chamber. each alpha ring makes up antechamber.
84
ClpAP function, structure
prokaryotic proteasome. ClpA ATP dependent caps. ClpP protease core
85
ubiquitin linked to ? on protein
lysine or alpha-NH2
86
ubiquitin bonds
protein linked to C-term Gly. adjacent ubiquitin linked between c-term gly and a lysine via isopeptide bond
87
cleaves isopeptide bond between ubiquitin monomers
isopeptidase. allows recycling
88
ubiquitin recycling general mechanism
E1 binds free Ub. Transfers to E2. E3 attaches to target protein via amide bond.
89
E1 binding Ub mechanism
Ub conjugated to Acyl-AMP via ATP hydrolysis. transferred to E1. form shigh energy thioester.
90
E3 Ub mechanism
E3 ligase binds protein noncovalently and transfers Ub covalently from E2. Forms isopeptide bond. There are many different E3 ligases that seek different proteins
91
half life of many proteins depends on this sequence
n-terminal sequence
92
destabilizing AA of N-term sequence that signal breakdown in prok and euk
FLY WRK
93
destabilizing AA of N-term sequence that signal breakdown in EUK
HATS
94
Effect of Asp or Glu on N-term
indirect destabilizing. Arg-tRNA transferase adds Arg that signals breakdown
95
Effect of Asn or Gln on N-term
Converted to Asp or Glu thentargetted for Arg addition for breakdown
96
Arg-tRNA transferase adds Arg that signals breakdown to these AA
Asp or Glu. Acidic
97
protein degradation sequence that does not involve N-rule
PEST sequence. any order. ubiquination by different set of enzymes
98
enzymes that ubiquinate PEST
Ubc1,4,5
99
HtrA dual function
chaperone at low temp, proteasome at high temp
100
HtrA catalytic triad
Asp-His-Ser
101
HtrA structure.
dimer of trimers. PDZ substrate recognition domains reach out. serine protease core.
102
caspases
cys+asp-ases. apoptosis. cleaves after Asp
103
metalloproteases
Zn requiring endopeptidase. Allow cells to invade be cleaving ECM proteins
104
viral proteinases
cleave viral polyprotein precursors. viral encoded. catalytic metal involved. site specific endopeptidase
105
induction
increased expression of genes in response to metabolite
106
activator
protein that binds operator to activate
107
inducer
small molecule that activates gene expression by binding repressor
108
constitutive gene
gene is always on at fixed rate
109
operon characteristics
genes involved in same pathway. identical transcription regulation. different translation regulation
110
ribosomal protein operons
operator is in mRNA
111
adaptation
change in expression in response to environmental change. small metabolite acts as inducer. decreases repressor affinity for DNA
112
promoters in lac operon
Pi for LacI (repressor), Po for Laz Z Y and A
113
Po operator. regulation?
LacO. LacI represses.
114
lac operator acronym
PIPOZYA. promoter-LacI-promoter-operator-lacZ-lacY-lacA
115
lac operon induction
allolactose acts as inducer and binds lacI (repressor)
116
uninduced vs induced lac operon protein levels
10 vs 50,000 copies of beta galactosidase and permease
117
common chemical analog of allolactose
IPTG
118
lac operator characteristics
three binding sites O1, O2 and O3. O1 is high affinity. Each are palindromic
119
Lac I structure and operator binding
dimer of dimers. each dimer binds different operator. interaction is helix-turn-helix
120
e. coli grown in? (glucose and lactose conc)
0.2 mg/ml glucose. 4 mg/ml lactose
121
why is beta-galactosidase gene not turned on until glucose is exhausted?
low glucose increases cAMP conc. activates CAP activator of Po
122
activation and derepression of lac operon
activation (CAP binding), derepression (induced lacI)
123
CAP activation of lac operon
as glucose is depleted, cAMP increases. binds CAP. CAP binds Po and recruits RNAP.
124
CAP global function
activator of alternative energy sources. lactose, galactose, arabinose...
125
CAP effect on DNA structure
introduces bend. protein side chan and DNA phosphate interaction
126
ara operon
turned on when arabinose is primary carbon source
127
turned on when arabinose is primary carbon source
ara operator
128
genes in the arabinose operator
B and A and D. araBAD
129
ara operon responds to ? and ?
CAP and araC
130
araC regulation of ara operon
araC gene makes araC. araC dimer forms at low arabinose and causes DNA looping over long distance upstream of ara operator. at high arabinose, araC binds CAP (no loop)
131
Trp operon encodes ?
leader sequence and 5 proteins involved in Trp synthesis
132
Trp repressor regulates ?
Trp operon AND trpR and aroH operons (autogenous)
133
attenuation of Trp operon requires?
alternative stem-loops. requires link between transcription and translation (prok). Consecutive Trp codons in mRNA leader
134
Trp operon attenuation at low Trp
slow ribosome (resulting from low Trp availability) allows base pairing between sequence 2 and 3. ribosome pausing is sequence 1 inhibits formation of terminator stem loop
135
Trp operon attenuation at high Trp
base pairing between sequence 3 and 4. ribosome translates through sequence 1 and 2. 3-4 stem loop inhibits RNAP. early transcription termination. fewer Trp enzymes
136
negative control induction
coinducer inactivates repressor
137
positive control induction
coinducer activates inducer
138
negative control repression
corepressor activates repressor
139
positive control repression
corepressor inactivates inducer
140
3 modes of transcription regulation
chromatin remodeling, gene arrangement, activation/repression
141
3 modes or translation regulation
alternative splicing, mRNA stability, translational control
142
eukaryote transcript processing in nucleus
5' cap (m7GpppG), 3' cleavage and polyadenylation, introns spliced
143
7 regulatory sites in eukaryote gene expression
chromatin, transcription control, RNA transport, RNA stability, translation control, protein processing
144
histone acetylation effect. enzymes
loosen nucleosome structure. HAT (acetylation) and HDAC (deacetylation)
145
heritable epigentic modification
histone modification as cellular memory
146
H3 Lys4 trimethylation effect on histone
gene activation
147
H3 Lys9 methylation effect on histone
gene repression
148
coinducer and corepressor effects on chromatin
coinducer or corepressor binds TF and has HAT or HDAC activity
149
CpG islands
regions of cysteine prone to methylation. occur near promoters. proteins bind that maintain inactive state
150
heterochromatin characteristics
DNA is methylated. histone tails not acetylated. histone tails methylated.
151
euchromatin characteristics
DNA not methylated, histone tails acetylated.
152
does histone tail acetylation expose DNA for transcription
not directly. chromatin remodeling complex causes ATP dependent conformational change
153
TATA box transcription factor
TBP
154
GC box transcription factor
SP1 (specificity protein)
155
helix-turn-helix DNA binding
c-term helix fits into major groove, N-term stabilizes C term
156
Zn finger motifs
C2H2 (form beta strand and alpha helix) or Cx (1 st helix dna binding, 2nd packs against first)
157
helix-turn-helix example
E. coli CAP, lacI
158
Zn finger example
TFIIIA
159
leucine zipper structure
28 AA w/ leucine every 7. y shaped dimer. arms bind DNA. stem holds together. homodimer recognizes symmetric site. heterodimer recognizes asymmetric.
160
Leu zipper example
Fos
161
activation of transcription by signal transduction example
Jak stat pathway response to interferons
162
PEPCK is stimulated for?
gluconeogenesis
163
clone DNA procedure
restriction endonuclease of DNA to be cloned and of plasmid. ligate. transform bacterial cell. select sells that contain plasmid
164
bioinformatics definition
detailed view of expression of all genes in any situation
165
DNA microarrays
determine individual identity of every mRNA in cell. quantify by extent of probe hybridization. synthesized DNA with unique sequences. fluorescent probe hybridizes. different tag for each population.
166
RNA deep sequencing
sequence (short stretches) each mRNA multiple times. quantify number of times found (abundance). determine isoform and type differences in mRNA
167
G1 phase
grow in size, catabolize nutrients, synthesize proteins,
168
S phase
replicate DNA
169
G2 phase
check and segregate cytoplasmic and nuclear contents
170
M phase
localize contents and divide
171
G0 phase
cells are still metabolizing but not replicating
172
cyclins degraded by
28S proteasome
173
cyclin D regulates __
G1/S transition
174
cyclin A regulates ___
S/G2 transition
175
cyclin B regulates ___
G2/M transition
176
CKIs
Cdk inhibitors
177
E2F function
induces proliferative genes
178
Rb function
tumor suppressor that binds E2F. Cells in G0 and G1 arrest
179
phosphorylate to inactive Rb
CyclinD/Cdk
180
epigenetic
stable change that alters DNA expression
181
angiogenesis
formation of new blood cells
182
formation of new blood cells
angiogenesis
183
stable change that alters DNA expression
epigenetic
184
p53 function
halts cell cycle when DNA is damaged. activates p21 gene and GADD45
185
p21 gene
cyclin/cdk inhibitor
186
GADD45 function
demethylation to inhibit growth
187
p53 expression in tumors
mutated in 50% of tumors
188
EGF receptor as an oncoprotein
epidermal growth factor. truncation makes constitutively active. referred to as ErbB protein. gene is amplified in 25% of breast cancers
189
multiple proto-oncogenes in ras pathway
ras, raf, fos, jun, myc
190
neurofibromatosis is due to ?. how?
Ras misregulation. NF-1 activates Ras innate GTPase activity. Ras-GDP no longer signals to Raf. mutations in NF-1 allows Ras-GTP to persist
191
overexpression of EIF4E or EIF4G
transformation and tumor formation
192
tamoxifen
antagonist of estrogen receptor
193
ER(+)
rely on estrogen for continued growth
194
means of circumventing apoptosis
mutate p53. upregulate Bcl2 (antiapoptotic). destroy capsase activity
195
release of ? assembles apoptosome
cytochrome C
196
Bcl-2 location and function
outer mitochondrial membrane protein
197
apoptosome function
releases caspases for apoptosis
198
extrinsic apoptosis signal
T-cell mediated
199
intrinsic apoptosis signal
GF depletion, irradiation
200
initiator caspases
8 and 9
201
executioner caspases
3, 6, and 7
202
Apaf1
inhibited by Bcl2. makes caspase 9. contains IRES. feedback loop
203
what was the conclusion of anfinsens experiment
all necessary information to determine 3D fold is in primary sequence
204
mitochondraa localization signal
amphipathic helix. positive charge on one side
205
nuclear localization signal
positively charged
206
SRP composition
ribonucleoprotein. 7sRNA (300nt) + 6 SRP proteins
207
SRP translation arrest mechanism
signal peptide binds SRP54. forms a kink involving srp68. Alu rotates to contact EFS (elongating factor binding site). Alu competes with EF. SRP stretches from peptide exit to the EFS.
208
26S proteasome 19S cap composition
15+ dif proteins, 6 ATPases, isopeptidase
209
where are 26S proteasomes located
nucleus and cytoplasm
210
step in ubiquitination that requires ATP
attachment of AMP to Ub C-term gly
211
HtrA catalytic triad
DHS
212
PDZ recognizes what?
protein substrate 3-4 C-term AAs
213
approximate # genes in c-elegans
17000
214
approximate # genes in humans
30000
215
jakstate activates gene transcription for ..
antiviral response, antitumor response
216
components of transcription complex
regulatory sequences, regulatory dna binding proteins, mediator proteins, promoter proximal elements, core promoter, basal transcription complex (RNApol and TFs)
