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BIS477 World of RNA > Mechanisms of translation regulation > Flashcards

Mechanisms of translation regulation Flashcards

1
Q

What is an ORF? (2)

A
  • Open reading frame
  • Protein coding region of DNA/RNA
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2
Q

What is a UTR? (2)

A
  • Untranslated region with regulatory properties
  • 5’ UTR and 3’ UTR around the protein encoding sequence in DNA/RNA which are transcribed but not translated into protein
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3
Q

What is the structure of the eukaryotic ribosome? (3)

A
  • 60S (peptidyl transferase centre - peptide bond formation)
  • 40S (decoding centre - mRNA binds)
  • Translation largely occurs at the interface between the 2 subunits
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4
Q

What are the 3 phases of the eukaryotic translation cycle?

A
  • Initiation
  • Elongation
  • Termination and recycling
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5
Q

What are the steps of translation initiation? (8)

A
  • Requires association and dissociation of numerous translation initiation factors (eIFs) throughout
  • Ternary complex forms (eIF2-GTP and Met-tRNAi) and binds to the 40S subunit forming the preinitiation complex (43S)
  • mRNA proximal to the 5’ cap is unwound via helicase activity of the eIF4F complex, circularises the mRNA and puts it in an active form
  • Active mRNA associates with the 43S preinitiation complex
  • 40S subunits scans along the 5’ UTR to look for AUG start codon
  • 48S initiation complex formation through eIF2-GTP hydrolysis to eIF2-GDP and dissociation from the 40S subunit
  • 60S ribosome subunit associates which requires eIF5b-GTP
  • eIF5b-GTP hydrolysis to eIF5b-GDP causes dissociation of remaining eIFs, leaving 80S initiation complex which is competent for elongation phase
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6
Q

What is the rate limiting step in translation? (2)

A
  • Initiation
  • Most subject to regulation
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7
Q

What are eIFs?

A

(Eukaryotic) translation initiation factors

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8
Q

What is Met-tRNAi?

A

Initiation tRNA bound to methionine because the start codon (AUG) encodes Met

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9
Q

What are the steps of translation elongation?

A

Insertion of the next aminoacyl-tRNAs and addition of amino acids to the polypeptide chain causes translocation of the ribosome along the mRNA

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10
Q

What are the steps of translation termination and recycling? (3)

A
  • Encounter a stop codon
  • Recruit translation termination and recycling factors
  • Causes disassembly of the ribosome and recycling of factors for the next round of translation
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11
Q

What are the stop codons? (3)

A
  • UAA
  • UAG
  • UGA
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12
Q

What is the ternary complex? (2)

A
  • Association between eIF2-GTP and Met-tRNAi during initiation
  • If not enough of this complex translation is blocked right from the start
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13
Q

What is eIF2? (2)

A
  • Eukaryotic initiation factor 2
  • GTPase
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14
Q

What is the preinitiation complex?

A

Ternary complex + 40S subunit

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15
Q

Why is global translation regulation important? (4)

A
  • Multiple mechanisms that can be manipulated in the initiation process
  • Important for rapid response to change in conditions e.g. stress
  • Can allow expression profile to be rapidly and reversibly changed by global up/downregulation
  • Quicker/easier to alter translation than transcription
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16
Q

What happens to translation under stress conditions?

A

Global downregulation

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17
Q

Which steps of translation initiation can be targeted to alter global translation? (2)

A
  • Interfere with mRNA activation through sequestration or cleavage of components of eIF4F
  • Decrease ternary complex formation through phosphorylation of eIF2
18
Q

How does phosphorylation of eIF2 prevent ternary complex formation? (5)

A
  • eIF2 GTPase has alpha, beta and gamma subunits
  • eIF2B GEF exchanges GDP for GTP
  • Alpha subunit of eIF2 is phosphorylated under stress (multiple possible kinases)
  • This binds and sequesters eIF2B so inhibits exchange of GDP to GTP
  • eIF2 needs to be GTP bound to form ternary complex so not enough ternary complex so global translation decreases
19
Q

What are examples of cellular stress? (7)

A
  • Amino acid starvation
  • UV irradiation
  • Viral infection
  • Osmotic shock
  • Heat shock
  • ER stress
  • Hypoxia
20
Q

Why do some genes bypass global downregulation of translation? (2)

A
  • Need to be able to express genes that can respond to damage so you can either repair the damage or undergo apoptosis
  • Therefore certain mRNAs are expressed more in response to eIF2 phosphorylation
21
Q

How are response genes that bypass global downregulation of translation regulated?

A

Elements in the 5’ UTRs

22
Q

Which elements of the 5’ UTR regulate bypass of global downregulation of translation? (2)

A
  • Upstream ORFs (uORFs)
  • Internal ribosome entry sites (IRES)
23
Q

What are uORFs? (2)

A
  • Small ORFs upstream from the ORF in the 5’ UTR which can influence expression of the downstream gene
  • Don’t always start with AUG codons, often near cognates
24
Q

What are IRESs? (4)

A
  • RNA motif structures in the 5’ UTR which can directly recruit 40S ribosome subunit
  • First discovered in viral genomes as they enable viruses to hijack the host cell’s translation machinery
  • Viral IRESs are classified into 4 types (I-IV)
  • Enable cap-INdependent translation so don’t need all the normal eIFs/machinery
25
Q

What elements in the mRNA 5’ UTR can regulate translation? (2)

A
  • Can enhance/inhibit expression of the downstream gene
  • E.g. uORF, IRES, binding sites for proteins/lncRNAs etc.
26
Q

What are the general features of uORFs? (3)

A
  • Require all the same translation machinery (cap-dependent translation)
  • > 50% eukaryotic mRNAs contain uORFs
  • Can have positive or negative influence on downstream gene expression
27
Q

How can uORFs have a positive effect on downstream gene expression?

A

Ribosome reinitiation

28
Q

What is ribosome reinitiation? (3)

A
  • Translation of uORF
  • Ribosome stops at the end of the uORF
  • 40S continues to scan down the mRNA and restarts translation at the proper uORF
29
Q

How can uORFs have a negative effect on downstream gene expression? (3)

A
  • Ribosome elongation stall
  • Ribosome dissociation
  • Ribosome translation past the CDS start codon
30
Q

What is ribosome elongation stall?

A

Ribosome stops during translation of the uORF and doesn’t continue

31
Q

What is ribosome dissociation?

A

Ribosome translates the uORF, falls off and doesn’t reinitiate

32
Q

What is ribosome translation past the CDS start codon?

A

uORF overlaps with the proper coding sequence so can’t get expression of the mRNA if the uORF is being expressed

33
Q

How can uORFs have no effect on downstream gene expression?

A

When the ribosome bypasses the uORF and just encodes the downstream gene

34
Q

What factors can influence the impact of the uORF on downstream gene expression? (2)

A
  • Sequence of the uORF, some are stronger/weaker
  • Conditions of the translation process can affect whether the components associate/dissociate/read-through etc
35
Q

What is ATF4? (2)

A
  • Transcription factor gene whose expression is controlled by 2 uORFs
  • Encourages transcription of stress response genes
36
Q

How is ATF4 controlled by uORFs? (4)

A
  • uORF1 is within the 5’ UTR, uORF2 overlaps the start site of ATF4 gene
  • ATF4 only needs to be expressed under stress
  • No stress = high eIF2-GTP, low eiF2alpha-P, lots of ternary complex so ribosome translates uORF1 and reinitiates at uORF2 = no ATF4 expression because uORF2 overlaps
  • Stress = low eIF2-GTP, high eiF2alpha-P, not much ternary complex, uORF1 is translated but takes longer to find more eIF2-GTP for ternary complex so re-initiation is delayed therefore uORF2 is skipped and ATF4 gene is translated
37
Q

How do cellular IRESs influence downstream gene expression? (4)

A
  • All cellular mRNAs are capped and can undergo cap-dependent translation
  • IRESs allow bypassing of normal translation initiation for cap-INdependent translation during global downregulation of translation e.g. during stress
  • Predicted that 10-15% of mammalian mRNAs contain IRESs
  • Few structural similarities make them hard to identify
38
Q

What are the structures of cellular IRESs? (3)

A
  • Cellular IRESs are less structured than viral
  • Complex RNA folds
  • Short sequence elements which directly bind ribosome
39
Q

How do cellular IRESs recruit ribosomes? (2)

A
  • Cellular IRESs use diverse modes of recruitment
  • Many IRESs require trans-acting factors to remodel the RNA for ribosome recruitment
40
Q

What is APAF1?

A

Pro-apoptotic factor involved in stress response

41
Q

How is APAF1 expression controlled by an IRES? (2)

A
  • 2 trans-acting factors UNR and NPTB bind in a hierarchical manner which remodel the RNA to create a 40S binding site, bypassing need for classical translation initiation
  • Allows APAF1 expression during global downregulation to enable apoptosis

BIS477 World of RNA (14 decks)

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