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CMB2001 - Eukaryotic Control of Gene Expression > Lectures 9-10: Post-transcriptional control of gene expression (pt2) > Flashcards

Lectures 9-10: Post-transcriptional control of gene expression (pt2) Flashcards

1
Q

What are the properties of prokaryotes 70S ribosome?

A

16S rRNA small subunit
23S + 5S rRNA large subunit

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2
Q

What are the properties of eukaryotes 80S ribosome?

A

18S rRNA small subunit
28S + 5.8S + 5S rRNA large subunit

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3
Q

What are the different loops within tRNA?

A

TΨC loop, Anticodon loop and D loop

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4
Q

What are the different types of bases contained in tRNA?

A

R - purine (G/A)
Y - pyrimidine (C,T or U)
Ψ - pseudouridine (most abundant port-transcriptionally modified nucleotide in cellular RNA)

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5
Q

How is pseudouridine produced?

A

Via an internal transglycosylation reaction using Ψ synthases

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6
Q

What is the 2 step reaction to produce aminoacyl groups?

A

1 - Amino acid activation - amino acid and ATP bind catalytic site, nucleophilic attack by α-carboxylic acid oxygen yielding aminoacyl-adenylate
2 - Hydroxyl group of adenine 76 of tRNA attacks the carbonyl carbon of the adenylate, forming aminoacyl-tRNA and AMP

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7
Q

How does translation elongation take place?

A

Charged tRNA is used which binds to the aminoacylated tRNA binding site
Peptide bond is formed between the peptides in the protein chain and the tRNA
A GTP and factor G (eIF2) complex is formed which allows the tRNA to move along to the polypeptide chain site
GTP is hydrolysed to GDP so the tRNA is then able to move along again and leave the ribosome via the exit site

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8
Q

What are they key factors of translation?

A

Peptide bond formation catalysed by the ribosome
RNA-catalysed event
Ribosomes contain many proteins - eukaryotic more complex
tRNA deliver the amino acid
tRNA are present in the P and A sites - expanding polypeptide chain is attached to P-site tRNA

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9
Q

What are PABs?

A

Poly(A) binding proteins

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10
Q

What is the process of eukaryotic translation initiation?

A

Small subunit of ribosome binds to the cap finding the first AUG (methionine)
This first base is usually found from the Kozak consensus sequence
Then the large subunit of the ribosome binds

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11
Q

How is mRNA circularised during translation initiation?

A

eIF4F complex allows the circulation by brining ribosomes ending =close to several other key translation factors

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12
Q

What are the different translation initiation factors?

A

eIF1A - 80S dissociation Met-tRNA binding to 40S
eIF3 - 80S dissociation binds to many other eIFs
eIF1 - AUG recognition
eIF2 - GTPase, Met-tRNA binding, binds eIF5
eIF5 - stimulates eIF2 GTPase GAP for eIF2

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13
Q

What is needed for 43S association during translation initiation?

A

Interactions eIF3 with eIF4G
RNA unwinding - most 5’ UTRs have at least some structure, eIF4F unwinds cap-proximal sequence

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14
Q

What are key things to remember about eukaryotic translation?

A

Eukaryotic mRNA contains cap and polyA tail
eIF4A complex binds cap - interacts with PAB circulating the mRNA
Small subunit - part of initiation complex, recruited to cap then scans to find AUG
Large subunit joins
Translocation required to more tRNA and mRNA through ribosome
Termination codon reached, translation stops and ribosome dissociates

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15
Q

What are the key points of translation regulation?

A

Function of eIF2B/ ternary complex formation
Formation of eIF4F
43S binding

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16
Q

What is eIF2 GEF?

A

eIF2 G-nucleotide exchange factor

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17
Q

What are the key properties of eIF2B?

A

Present at a lower level than eIF2
eIF2B activity governs level of active eIF2-GTP and overall initiation rate
eIF2B activity is down-regulated in response to stresses (e.g. viral infection, amino-acid deprivation and endoplasmic reticulum stress)
Regulation through phosphorylation of eIF2, competitive inhibitor of eIF2B
Consequence: generation of ternary complex is impaired, translation initiation of mRNA is reduced

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18
Q

What happens if no eIF4A complex is formed?

A

No initiation of translation

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19
Q

What are the factors of eIF2?

A

eIF2 - 3 subunits α, β and γ
eIF2α - phosphorylated on Ser51 by PKR, PERK, GCN2 and HRI (kinases)
eIF2β - binds eIF2B and eIF5
eIF2γ - GTPase and Met-tRNA

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20
Q

What is a key regulatory step of the eIF2 cycle?

A

Since eIF2 > eIF2B, small amount of eIF2 phosphorylation can decrease ternary complex levels substantially

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21
Q

What are the different eIF2 kinases?

A

PKR, PERK, GCN2 & HRI

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22
Q

How is PKR eIF2 kinase activated?

A

By double stranded RNA (viral infection)

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23
Q

What is the function of PERK eIF2 kinase?

A

It is a mediator of the unfolded protein response

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24
Q

What is the function of HRI eIF2 kinase?

A

It links globing availability to protein synthesis (RBC biogenesis)

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25
Q

What is the function of GCN2 eIF2 kinase?

A

It is a regulator of translation in response to amino acid availability

26
Q

What are the key factors of the eIF2 kinases?

A

They dimerise under stimulatory conditions
Then autophosphorylate and act on substrate

27
Q

How does PKR eIF2 kinase act as an antiviral defence strategy?

A

PKR expression typically low
Increases when cells are exposed to interferons
Interferons are produced and released by cells infected by viruses
dsRNA binding leads to reproduction of RNA viruses generating dsRNA
When PKR binds to dsRNA it dimerises and is activated

28
Q

How is iron metabolism regulated?

A

Stored as ferritin
Exported as ferroportin
Used in metabolic utilisation to form Fe-proteins

29
Q

What are UTRs?

A

Un-translated regions

30
Q

Where can UTRs be found?

A

At the 5’ and 3’ of mRNA

31
Q

What is the function of UTRs?

A

To regulate and stabilise translation

32
Q

What are IREs?

A

Iron response elements

33
Q

What are IREs used for?

A

They are found in hairpin loops with a conserved sequence and bulge within the stem
Found in the 5’ or 3’ UTRs of iron regulated mRNAs
Bound by iron regulatory proteins IRP1 and IRP2

34
Q

What happens to iron storage at low levels of iron?

A

Translation is repressed as IRP1/2 binds containing 5’/3’ UTRs

35
Q

What happens to iron transport at low levels of iron?

A

mRNA is stabilised as IRP1/2 bind at the 3’ end

36
Q

What happens to iron storage at high levels of iron?

A

Translation is activated as IRP2 is degraded and IRP1 is blocked meaning they are unable to bind

37
Q

What happens to iron transport at high levels of iron?

A

mRNA is regraded are RNase breaks the mRNA down and IRP1 is blocked and IRP2 is also degraded

38
Q

What is IRP1?

A

A bifunctional protein as it can for c-aconitase by adding iron and Fe-S cluster assembly enzymes

39
Q

Why would RNA be degraded?

A

Damage
Incorrectly transcribed/processed
Control gene expression

40
Q

What is an example of control of gene expression using mRNA?

A

Casein mRNA
Expressed in mammary gland
Increases ~70 fold on stimulation by prolactin
Transcription only increases ~2 fold
Half-life increases dramatically as poly(A) tail is increased 3’ UTR stabilises

41
Q

How do eukaryotic mRNAs behave during translation?

A

They are circular which allows:
Monitoring of mRNA integrity (lost cap or poly(A) tail)
Brings ribosomes ends translation close to AUG

42
Q

What is deadenylation-dependent decay?

A

CCR4-NOT complex or PARN deadenylates the mRNA
3’ → 5’
Exosome unwinds the exposed mRNA
m7G cap is removed by DcpS
5’ → 3’
mRNA exposed so decapping takes place using the enzyme DCP2
XRN1 is then used to degrade the mRNA

43
Q

What are the different decapping enzymes?

A

DCP1 and DCP2

44
Q

What are the different endonucleases used to degrade mRNA?

A

Argonaute
Swt1
Smg6

45
Q

What are the different deadenylases used to degrade mRNA?

A

CCR/NOT complex

46
Q

What are the different nuclease activities during mRNA degradation?

A

RRP6 and RRP44

47
Q

What is XRN1?

A

A 5’ to 3’ exonuclease
Involved in RNA turnover and processing
Transcription termination
Functions after decapping

48
Q

What causes deadenylation-dependent decay?

A

AU-rich element (ARE)
Nonsense codon
c-fos major codon determinant
miRNA recognition site with miRNA bound

49
Q

What is nonsense mediated decay (NMD)?

A

Mistakes in mRNA meaning it gets degraded
Premature stop codons

50
Q

What can result from premature stop codons(PSC)?

A

Errors in:
Transcription
Splicing
Editing
Polyadenylation
Mutations

51
Q

How are 1/3 of inherited disorders caused?

A

By nonsense/frameshift mutations that introduce premature stop codons (PSC)

52
Q

How can nonsense mediated decay targets be determined?

A

if there are sites between exon junction complexes where there are more than 55 nucleotides

53
Q

What is the mechanism when nonsense mediated decay(NMD) is detected?

A

First round of translation, exon junction complexes (EJCs) are removed from mRNA by the ribosome
When ribosome reaches the premature stop codon a junction remains downstream so specific factors part of the junction are recruited to interact with RNA degradation machinery

54
Q

What are the different RNAs and the complex?

A

RNAi - RNA interference
siRNA - small inhibitory RNA
miRNA - micro RNA
RISC - RNA induced silencing complex

55
Q

What are the differences between siRNA and miRNA?

A

Both 21-23 nucleotides
miRNA - imperfect complementary to target RNA
miRNA - key gene regulatory mechanism
miRNA - leads to block in translation
siRNA - leads to degradation of target RNA
siRNA - viral defence mechanism
siRNA - prefect complementary to target RNA

56
Q

How is miRNA produced?

A

Pri-miRNA formed in hairpin loops
↓drosha
pre-miRNA
↓exportin 5
miRNA nucleus to cytoplasm
↓dicer
miRNA (duplex)

57
Q

How is siRNA produced?

A

dsDNA
↓dicer
siRNA duplex

58
Q

How is miRNA assembled into RISC?

A

miRNA duplex
↓unwinds
asymmetric RISC assembly
↓
Translational repression at ribosome

59
Q

How is siRNA assembled into RISC?

A

siRNA duplex
↓unwinds
siRNA
↓+some miRNA
RISC
↓target mRNA
mRNA cleavage

60
Q

How do 3’ UTR lengths change?

A

During embryonic development they get longer
mRNA in proliferating cells tend to have shorter 3’ UTRs
Driven by use of alternate polyadenylation sites
Longer 3’ UTR has more possibility of binding sites for miRNAs

CMB2001 - Eukaryotic Control of Gene Expression (4 decks)

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