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Mol Biochem > Exam 4 > Flashcards

Exam 4 Flashcards

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1
Q

initiator tRNA in prokaryotes

A

n-formyl-Met-tRNA^Met. only prokaryote is formylated

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2
Q

what part of prokaryotic ribosome binds to S/D

A

3’ end of 16s RNA

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3
Q

initiation codon is ? and occasionally ? in prokaryotes

A

AUG.. GUG

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4
Q

adds formyl group to initiator tRNA

A

formyl transferase

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5
Q

three prokarotic initiation factors

A

IF1, IF2 and IF3

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6
Q

IF-1

A

bonds 30s, prevents tRNA binding A site

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7
Q

IF-2

A

binds fMet-tRNA and delivers to P site. interacts with IF-1-30s. forms GTP dependent ternary complex

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8
Q

IF-3

A

binds to E site on 30s. prevents 50s joining in absence of mRNA. Aligns mRNA on 30s. Assists IF-2 placement

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9
Q

prokaryote: initiation. 3 steps

A

30S preparation > ternary complex/mRNA joining > 50s joining

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10
Q

prokaryote 30S prep details

A

30S and 50S dissociate. IF 1 and 3 binds 30S at the A and E site. IF-3 prevents 50S joining.

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11
Q

prokaryote ternary complex/mRNA joining detail

A

IF-2 binds fMET-tRNA and GTP to form ternary complex. ternary complex and mRNA bind to 30S-IF1-IF3. IF-2 delivers fMET-tRNA. S/D aligns 16S (of 30S)

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12
Q

prokaryote 50S joining detail

A

GTP hydrolyzed on 50S joining. IF’s dissociate. 70S initiation complex is formed

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13
Q

prokaryote elongation factors

A

EF-Tu, EF-Ts, EF-G

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14
Q

prokaryote EF-Tu

A

GTP dependent ternary complex with tRNA. delivers tRNA to 70s A site. GTP hydrolysis and release.

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15
Q

prokaryote EF-Ts

A

GTP exchange for GDP on EF-Tu

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16
Q

prokaryote EF-G

A

ribosome translocation. Uses GTP hydrolysis

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17
Q

Steps in prokaryote elongation

A

EF-Tu-GTP delivers charged tRNAs > peptide formed > EF-G promotes ribosome translocation > GTP exchange

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18
Q

prokaryote charged tRNA delivery detail

A

ternary complex EF-Tu-GTP-tRNA delivers tRNA to A site. GTP hydrolyzed. GDP remains bound to be recycled.

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19
Q

slow step in elongation

A

accomodation, positioning tRNAs in P and A sites for bond formation

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20
Q

prokaryote peptide bond form detail

A

A2451 catalyzes A site AA nucleophilic attack of P site

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21
Q

prokaryote ribosome translocation detail

A

EF-G-GTP wedges into A site via interaction with 50s. triggers GTP hydrolysis. 30S ratchets relative to 50S. 70S ribosome moves 1 codon. EF-G, GDP, and Pi released

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22
Q

EF-G structure mimics ___

A

EF-Tu + tRNA

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23
Q

rank prokaryote replication, transcription and translation rates

A

replication&raquo_space;» transcription = translation

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24
Q

prokaryote GTP exchange detail

A

EF-Ts binds EF-Tu displacing GDP. GTP binds complex to displace EF-TS to form EF-Tu-GTP. EF-Tu-GTP binds new tRNA

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25
prokaryote RF-1
binds to A site with UAA or UAG codon
26
prokaryote RF-2
binds to A site with UAA or UGA
27
prokaryote RF-3
disociation of RF 1 and 2 from ribosome
28
prokaryote RRF
dissociates mRNA and tRNA
29
four prokaryote release factors
RF-1, RF-2, RF-3, RRF
30
what is unique about RRF
looks like tRNA but binds orthogonally across A site
31
eukaryotic initiation factors
eIF1-6
32
eIF1 and 1A
stabilize 43S and 48S
33
eIF2 and 2B
met-tRNA binding to 40S, GTP dependent ternary. GTP exchange by 2B
34
eIF3
promotes 40s binding to ternary and mRNA
35
eIF4
multisubunit A-G + PABP. RNA helicase, binds mRNA cap and tail
36
eIF5 and 5B
stimulates eIF2 GTPase, release initiation factors. joins 60S to 48S
37
eIF6
binds 60S to dissociate 40S
38
Overall eukaryote initiation steps
40/60S dissociation > 40S activation > recruit mRNA > join complexes > scan > AUG recognitio > factor release > 60S joining > eIF2 recycling
39
eukaryote 40/60S dissociation > 40S activation details
80S dissociates into 40 and 60s by eIF6 binding 60S. ternary complex forms (eIF2-GTP-met-tRNA). ternary joins eIF3 and 1A on 40S to form 43S
40
eukaryote recruit mRNA > join complexes detail
usually rate limiting. eIF4 recruits mRNA by recognizing cap and tail. unwinds secondary structure. joins mRNA to 43S to form 48S. addition of 60S not regulated.
41
eIF4E function and structure
binds directly to mRNA cap. palm up hand. cap is between finger and thumb. mRNA across palm dorsal helices. tryptophans stabilize unique methyl cap
42
complex that circularizes mRNA
eIF4
43
? complex scans 5' UTR to AUG codon
48S
44
what happens once 48S recognizes AUG and Kozak context
eIF5 joins, GTP hydrolysis > complex clamps. eIF factors released
45
difference between S/D and kozak recognition
30S binds directly to S/D whereas no BP between 48S and kozak
46
eukaryote 60s joining and factor recycling detail
eIF5B catalyzes 60S joining. eIF2 brings aminoacyl tRNA to A site. eIF2B exhanges GTP for eIF2 GDP
47
eIF2 phosphorylation effect
binds irreversibly to eIF2B
48
eukaryote elongation factors
eEF1alpha, eEF1betagamma, eEF2
49
eEF1alpha
GTP dependent ternary, delivers AAtRNA to 80S A site. GTP hydrolysis and release.
50
eEF1betagamma
GTP exchange for GDP on eEF1alpha
51
eEF2
ribosome translocation by GTP hydrolysis
52
overall steps in eukaryote elongation
eEF1alpha delivers charged tRNAs > eEF2 aids ribosome translocation > eEF1betagamma exhanges GTP
53
eukaryote termination detail
eRF1-GTP recognizes A site with stop. peptidyl transferase hydrolyzes aminoacyl with water to release protein. eRF3 GTPase eRF1-GTP hydrolysis to GDP
54
how might eukaryotes link termination to initiation
eRF3 binds PABP. eIF4 may remain bound to mRNA
55
how does eRF1 trick ribosome into hydrolyzing aminoacyl
mimics tRNA structure but no AA present
56
eukaryote translation energy cost
4 ATP / AA + unwinding energy... 90% of metabolic energy
57
how are polysomes fractionated
sucrose gradient
58
eukaryote energy consuming steps
eIF4A helicase unwinding. 2n ATP tRNA charging. 2n GTP AA delivery and translocation.
59
how to watch mRNA enter polysomes
remove endogenous mRNA w/ nuclease. add purified 3H UTP-labelled histone mRNA. remove aliquots at time points to ice. sucrose gradient to resolve aliquots
60
initiation gets faster/slower as polysome builds
faster
61
translational efficiency
measure of number of ribosome on mRNA. protein made/mRNA*min
62
how to measure translation efficiency by sucrose gradient
sucrose gradient, isolate fractions, norther blot or real time PCR. efficiently translated mRNA will have a higher %mRNA in heavier fractions where ribosomes are more saturating
63
how to measure translation efficiency by pulse chase
pulse: temporarily introduce and remove labelled 35SMet to medium to measure uptake rate. chase: allow futher incorporation of unlabelled Met and measure any decrease in radioactivity.
64
lodish model
discriminating control by initiation factors (ie. eIF4). slowed by high secondary structure and bound RNPs
65
initiation regulated at two phases
ternary complex formation (eIF2+GTP+tRNAMet) and mRNA recuitment (eIF4+mRNA)
66
regulation of ternary complex formation (eIF2)
phosphorylation of eIF2 causes sequestration by eIF2B slowing initiation
67
regulation of mRNA recruitment (eiF4)
slowed by repressed or sequestered mRNA. activated by phosphorylation of 4EBP to release eIF4E. activated by phosphorylation of eIF4E and eIF4G.
68
affect of phosphorylated eIF2 on yeast GCN4
scanning intiation complex skips early AUGs and hits later.. encodes for GCN4
69
eIF2 kinases (4)
HRI, GCN2, PKR, PERK
70
HRI
eIF2 kinase activated by insufficient heme
71
GCN2
eIF2 kinase activated by AA starvation
72
PKR
eIF2 kinase activated to prevent viral proliferation
73
PERK
eIF2 kinase activated during ER stress
74
cap-dependent recognition requires
eIF4E-eIF4G complex. 4EBP competes with 4G
75
how do miRNA regulate mRNA
directs repression
76
how to siRNA regulate mRNA
directs degradation via decapping or de adenylation
77
three types of mRNA recruitment
via cap, tail, or IRES
78
what is IRES
internal ribosome entry site. eIF4G can bind directly
79
regulatory protease effect on eIF4 complex
cleaves at hinge. removes cap and tail recognition abilities. still active for cap independent. beneficial for viral mRNA that encode IRISes
80
stimulates cap dependent GLUT4 and cyclin D1 translation
insulin
81
cellular iron homeostasis
hairpin sequence (IRE) binds IRP1. blocks eIF4E/4G binding
82
IRP-1 affect on transferrin
blocks degradation
83
which phases are targeted by inhibitors
initiation and elongation
84
tetracycline
binds prokaryotic 30S. blocks tRNA binding A site
85
erythromycin
blocks prokaryotic 50S subunit tunnel. short products
86
chloramphenicol
binds prokaryotic 50S. blocks tRNA binding A site
87
streptomycin, neomycin, gentamycin
binds prokaryotic 30S, causes codon misreading. leads to misincorporation. can circumvent nonsense
88
puromycin
mimics tyrosyl tRNA. causes premature chain termination. prokaryote and eukaryote. binds A site
89
cycloheximide
binds eukaryote 80S. freezes ribosomes on mRNA
90
fusidic acid
blocks EF-G function
91
edeine
prevents AUG recognition
92
m7GTP
competes with mRNA cap for eIF4E binding
93
GMPPNP
non hydrolyzable GTP prevents eIF2 function
94
diptheria
ADP ribosylates eEF2
95
ricin inactivates __ by ?
60S by removing single adenine base. blocks eEF1 and 2 function
96
affect of EDTA on ribosomes
dissociates subunits
97
ribo-seq
detailed analysis of ribosome density | on individual RNAs

Mol Biochem (6 decks)

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