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

Question 1

What are the properties of prokaryotes 70S ribosome?

Answer 1

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

Question 2

What are the properties of eukaryotes 80S ribosome?

Answer 2

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

Question 3

What are the different loops within tRNA?

Answer 3

TΨC loop, Anticodon loop and D loop

Question 4

What are the different types of bases contained in tRNA?

Answer 4

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

Question 5

How is pseudouridine produced?

Answer 5

Via an internal transglycosylation reaction using Ψ synthases

Question 6

What is the 2 step reaction to produce aminoacyl groups?

Answer 6

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

Question 7

How does translation elongation take place?

Answer 7

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

Question 8

What are they key factors of translation?

Answer 8

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

Question 9

What are PABs?

Answer 9

Poly(A) binding proteins

Question 10

What is the process of eukaryotic translation initiation?

Answer 10

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

Question 11

How is mRNA circularised during translation initiation?

Answer 11

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

Question 12

What are the different translation initiation factors?

Answer 12

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

Question 13

What is needed for 43S association during translation initiation?

Answer 13

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

Question 14

What are key things to remember about eukaryotic translation?

Answer 14

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

Question 15

What are the key points of translation regulation?

Answer 15

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

Question 16

What is eIF2 GEF?

Answer 16

eIF2 G-nucleotide exchange factor

Question 17

What are the key properties of eIF2B?

Answer 17

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

Question 18

What happens if no eIF4A complex is formed?

Answer 18

No initiation of translation

Question 19

What are the factors of eIF2?

Answer 19

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

Question 20

What is a key regulatory step of the eIF2 cycle?

Answer 20

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

Question 21

What are the different eIF2 kinases?

Answer 21

PKR, PERK, GCN2 & HRI

Question 22

How is PKR eIF2 kinase activated?

Answer 22

By double stranded RNA (viral infection)

Question 23

What is the function of PERK eIF2 kinase?

Answer 23

It is a mediator of the unfolded protein response

Question 24

What is the function of HRI eIF2 kinase?

Answer 24

It links globing availability to protein synthesis (RBC biogenesis)

Question 25

What is the function of GCN2 eIF2 kinase?

Answer 25

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

Question 26

What are the key factors of the eIF2 kinases?

Answer 26

They dimerise under stimulatory conditions Then autophosphorylate and act on substrate

Question 27

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

Answer 27

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

Question 28

How is iron metabolism regulated?

Answer 28

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

Question 29

What are UTRs?

Answer 29

Un-translated regions

Question 30

Where can UTRs be found?

Answer 30

At the 5' and 3' of mRNA

Question 31

What is the function of UTRs?

Answer 31

To regulate and stabilise translation

Question 32

What are IREs?

Answer 32

Iron response elements

Question 33

What are IREs used for?

Answer 33

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

Question 34

What happens to iron storage at low levels of iron?

Answer 34

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

Question 35

What happens to iron transport at low levels of iron?

Answer 35

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

Question 36

What happens to iron storage at high levels of iron?

Answer 36

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

Question 37

What happens to iron transport at high levels of iron?

Answer 37

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

Question 38

What is IRP1?

Answer 38

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

Question 39

Why would RNA be degraded?

Answer 39

Damage Incorrectly transcribed/processed Control gene expression

Question 40

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

Answer 40

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

Question 41

How do eukaryotic mRNAs behave during translation?

Answer 41

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

Question 42

What is deadenylation-dependent decay?

Answer 42

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

Question 43

What are the different decapping enzymes?

Answer 43

DCP1 and DCP2

Question 44

What are the different endonucleases used to degrade mRNA?

Answer 44

Argonaute Swt1 Smg6

Question 45

What are the different deadenylases used to degrade mRNA?

Answer 45

CCR/NOT complex

Question 46

What are the different nuclease activities during mRNA degradation?

Answer 46

RRP6 and RRP44

Question 47

What is XRN1?

Answer 47

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

Question 48

What causes deadenylation-dependent decay?

Answer 48

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

Question 49

What is nonsense mediated decay (NMD)?

Answer 49

Mistakes in mRNA meaning it gets degraded Premature stop codons

Question 50

What can result from premature stop codons(PSC)?

Answer 50

Errors in: Transcription Splicing Editing Polyadenylation Mutations

Question 51

How are 1/3 of inherited disorders caused?

Answer 51

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

Question 52

How can nonsense mediated decay targets be determined?

Answer 52

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

Question 53

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

Answer 53

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

Question 54

What are the different RNAs and the complex?

Answer 54

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

Question 55

What are the differences between siRNA and miRNA?

Answer 55

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

Question 56

How is miRNA produced?

Answer 56

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

Question 57

How is siRNA produced?

Answer 57

dsDNA ↓dicer siRNA duplex

Question 58

How is miRNA assembled into RISC?

Answer 58

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

Question 59

How is siRNA assembled into RISC?

Answer 59

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

Question 60

How do 3' UTR lengths change?

Answer 60

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