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Mol Biochem > Exam 4 > Flashcards

Exam 4 Flashcards

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
Q

prokaryote RF-1

A

binds to A site with UAA or UAG codon

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26
Q

prokaryote RF-2

A

binds to A site with UAA or UGA

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27
Q

prokaryote RF-3

A

disociation of RF 1 and 2 from ribosome

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28
Q

prokaryote RRF

A

dissociates mRNA and tRNA

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29
Q

four prokaryote release factors

A

RF-1, RF-2, RF-3, RRF

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30
Q

what is unique about RRF

A

looks like tRNA but binds orthogonally across A site

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31
Q

eukaryotic initiation factors

A

eIF1-6

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32
Q

eIF1 and 1A

A

stabilize 43S and 48S

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33
Q

eIF2 and 2B

A

met-tRNA binding to 40S, GTP dependent ternary. GTP exchange by 2B

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34
Q

eIF3

A

promotes 40s binding to ternary and mRNA

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35
Q

eIF4

A

multisubunit A-G + PABP. RNA helicase, binds mRNA cap and tail

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36
Q

eIF5 and 5B

A

stimulates eIF2 GTPase, release initiation factors. joins 60S to 48S

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37
Q

eIF6

A

binds 60S to dissociate 40S

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38
Q

Overall eukaryote initiation steps

A

40/60S dissociation > 40S activation > recruit mRNA > join complexes > scan > AUG recognitio > factor release > 60S joining > eIF2 recycling

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39
Q

eukaryote 40/60S dissociation > 40S activation details

A

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
Q

eukaryote recruit mRNA > join complexes detail

A

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
Q

eIF4E function and structure

A

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
Q

complex that circularizes mRNA

A

eIF4

43
Q

? complex scans 5’ UTR to AUG codon

A

48S

44
Q

what happens once 48S recognizes AUG and Kozak context

A

eIF5 joins, GTP hydrolysis > complex clamps. eIF factors released

45
Q

difference between S/D and kozak recognition

A

30S binds directly to S/D whereas no BP between 48S and kozak

46
Q

eukaryote 60s joining and factor recycling detail

A

eIF5B catalyzes 60S joining. eIF2 brings aminoacyl tRNA to A site. eIF2B exhanges GTP for eIF2 GDP

47
Q

eIF2 phosphorylation effect

A

binds irreversibly to eIF2B

48
Q

eukaryote elongation factors

A

eEF1alpha, eEF1betagamma, eEF2

49
Q

eEF1alpha

A

GTP dependent ternary, delivers AAtRNA to 80S A site. GTP hydrolysis and release.

50
Q

eEF1betagamma

A

GTP exchange for GDP on eEF1alpha

51
Q

eEF2

A

ribosome translocation by GTP hydrolysis

52
Q

overall steps in eukaryote elongation

A

eEF1alpha delivers charged tRNAs > eEF2 aids ribosome translocation > eEF1betagamma exhanges GTP

53
Q

eukaryote termination detail

A

eRF1-GTP recognizes A site with stop. peptidyl transferase hydrolyzes aminoacyl with water to release protein. eRF3 GTPase eRF1-GTP hydrolysis to GDP

54
Q

how might eukaryotes link termination to initiation

A

eRF3 binds PABP. eIF4 may remain bound to mRNA

55
Q

how does eRF1 trick ribosome into hydrolyzing aminoacyl

A

mimics tRNA structure but no AA present

56
Q

eukaryote translation energy cost

A

4 ATP / AA + unwinding energy… 90% of metabolic energy

57
Q

how are polysomes fractionated

A

sucrose gradient

58
Q

eukaryote energy consuming steps

A

eIF4A helicase unwinding. 2n ATP tRNA charging. 2n GTP AA delivery and translocation.

59
Q

how to watch mRNA enter polysomes

A

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
Q

initiation gets faster/slower as polysome builds

A

faster

61
Q

translational efficiency

A

measure of number of ribosome on mRNA. protein made/mRNA*min

62
Q

how to measure translation efficiency by sucrose gradient

A

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
Q

how to measure translation efficiency by pulse chase

A

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
Q

lodish model

A

discriminating control by initiation factors (ie. eIF4). slowed by high secondary structure and bound RNPs

65
Q

initiation regulated at two phases

A

ternary complex formation (eIF2+GTP+tRNAMet) and mRNA recuitment (eIF4+mRNA)

66
Q

regulation of ternary complex formation (eIF2)

A

phosphorylation of eIF2 causes sequestration by eIF2B slowing initiation

67
Q

regulation of mRNA recruitment (eiF4)

A

slowed by repressed or sequestered mRNA. activated by phosphorylation of 4EBP to release eIF4E. activated by phosphorylation of eIF4E and eIF4G.

68
Q

affect of phosphorylated eIF2 on yeast GCN4

A

scanning intiation complex skips early AUGs and hits later.. encodes for GCN4

69
Q

eIF2 kinases (4)

A

HRI, GCN2, PKR, PERK

70
Q

HRI

A

eIF2 kinase activated by insufficient heme

71
Q

GCN2

A

eIF2 kinase activated by AA starvation

72
Q

PKR

A

eIF2 kinase activated to prevent viral proliferation

73
Q

PERK

A

eIF2 kinase activated during ER stress

74
Q

cap-dependent recognition requires

A

eIF4E-eIF4G complex. 4EBP competes with 4G

75
Q

how do miRNA regulate mRNA

A

directs repression

76
Q

how to siRNA regulate mRNA

A

directs degradation via decapping or de adenylation

77
Q

three types of mRNA recruitment

A

via cap, tail, or IRES

78
Q

what is IRES

A

internal ribosome entry site. eIF4G can bind directly

79
Q

regulatory protease effect on eIF4 complex

A

cleaves at hinge. removes cap and tail recognition abilities. still active for cap independent. beneficial for viral mRNA that encode IRISes

80
Q

stimulates cap dependent GLUT4 and cyclin D1 translation

A

insulin

81
Q

cellular iron homeostasis

A

hairpin sequence (IRE) binds IRP1. blocks eIF4E/4G binding

82
Q

IRP-1 affect on transferrin

A

blocks degradation

83
Q

which phases are targeted by inhibitors

A

initiation and elongation

84
Q

tetracycline

A

binds prokaryotic 30S. blocks tRNA binding A site

85
Q

erythromycin

A

blocks prokaryotic 50S subunit tunnel. short products

86
Q

chloramphenicol

A

binds prokaryotic 50S. blocks tRNA binding A site

87
Q

streptomycin, neomycin, gentamycin

A

binds prokaryotic 30S, causes codon misreading. leads to misincorporation. can circumvent nonsense

88
Q

puromycin

A

mimics tyrosyl tRNA. causes premature chain termination. prokaryote and eukaryote. binds A site

89
Q

cycloheximide

A

binds eukaryote 80S. freezes ribosomes on mRNA

90
Q

fusidic acid

A

blocks EF-G function

91
Q

edeine

A

prevents AUG recognition

92
Q

m7GTP

A

competes with mRNA cap for eIF4E binding

93
Q

GMPPNP

A

non hydrolyzable GTP prevents eIF2 function

94
Q

diptheria

A

ADP ribosylates eEF2

95
Q

ricin inactivates __ by ?

A

60S by removing single adenine base. blocks eEF1 and 2 function

96
Q

affect of EDTA on ribosomes

A

dissociates subunits

97
Q

ribo-seq

A

detailed analysis of ribosome density

on individual RNAs

Mol Biochem (6 decks)

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