Mechanisms of translation regulation Flashcards
What is an ORF? (2)
- Open reading frame
- Protein coding region of DNA/RNA
What is a UTR? (2)
- 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
What is the structure of the eukaryotic ribosome? (3)
- 60S (peptidyl transferase centre - peptide bond formation)
- 40S (decoding centre - mRNA binds)
- Translation largely occurs at the interface between the 2 subunits
What are the 3 phases of the eukaryotic translation cycle?
- Initiation
- Elongation
- Termination and recycling
What are the steps of translation initiation? (8)
- 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
What is the rate limiting step in translation? (2)
- Initiation
- Most subject to regulation
What are eIFs?
(Eukaryotic) translation initiation factors
What is Met-tRNAi?
Initiation tRNA bound to methionine because the start codon (AUG) encodes Met
What are the steps of translation elongation?
Insertion of the next aminoacyl-tRNAs and addition of amino acids to the polypeptide chain causes translocation of the ribosome along the mRNA
What are the steps of translation termination and recycling? (3)
- 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
What are the stop codons? (3)
- UAA
- UAG
- UGA
What is the ternary complex? (2)
- Association between eIF2-GTP and Met-tRNAi during initiation
- If not enough of this complex translation is blocked right from the start
What is eIF2? (2)
- Eukaryotic initiation factor 2
- GTPase
What is the preinitiation complex?
Ternary complex + 40S subunit
Why is global translation regulation important? (4)
- 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
What happens to translation under stress conditions?
Global downregulation
Which steps of translation initiation can be targeted to alter global translation? (2)
- Interfere with mRNA activation through sequestration or cleavage of components of eIF4F
- Decrease ternary complex formation through phosphorylation of eIF2
How does phosphorylation of eIF2 prevent ternary complex formation? (5)
- 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
What are examples of cellular stress? (7)
- Amino acid starvation
- UV irradiation
- Viral infection
- Osmotic shock
- Heat shock
- ER stress
- Hypoxia
Why do some genes bypass global downregulation of translation? (2)
- 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
How are response genes that bypass global downregulation of translation regulated?
Elements in the 5’ UTRs
Which elements of the 5’ UTR regulate bypass of global downregulation of translation? (2)
- Upstream ORFs (uORFs)
- Internal ribosome entry sites (IRES)
What are uORFs? (2)
- 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
What are IRESs? (4)
- 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
What elements in the mRNA 5’ UTR can regulate translation? (2)
- Can enhance/inhibit expression of the downstream gene
- E.g. uORF, IRES, binding sites for proteins/lncRNAs etc.
What are the general features of uORFs? (3)
- Require all the same translation machinery (cap-dependent translation)
- > 50% eukaryotic mRNAs contain uORFs
- Can have positive or negative influence on downstream gene expression
How can uORFs have a positive effect on downstream gene expression?
Ribosome reinitiation
What is ribosome reinitiation? (3)
- 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
How can uORFs have a negative effect on downstream gene expression? (3)
- Ribosome elongation stall
- Ribosome dissociation
- Ribosome translation past the CDS start codon
What is ribosome elongation stall?
Ribosome stops during translation of the uORF and doesn’t continue
What is ribosome dissociation?
Ribosome translates the uORF, falls off and doesn’t reinitiate
What is ribosome translation past the CDS start codon?
uORF overlaps with the proper coding sequence so can’t get expression of the mRNA if the uORF is being expressed
How can uORFs have no effect on downstream gene expression?
When the ribosome bypasses the uORF and just encodes the downstream gene
What factors can influence the impact of the uORF on downstream gene expression? (2)
- Sequence of the uORF, some are stronger/weaker
- Conditions of the translation process can affect whether the components associate/dissociate/read-through etc
What is ATF4? (2)
- Transcription factor gene whose expression is controlled by 2 uORFs
- Encourages transcription of stress response genes
How is ATF4 controlled by uORFs? (4)
- 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
How do cellular IRESs influence downstream gene expression? (4)
- 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
What are the structures of cellular IRESs? (3)
- Cellular IRESs are less structured than viral
- Complex RNA folds
- Short sequence elements which directly bind ribosome
How do cellular IRESs recruit ribosomes? (2)
- Cellular IRESs use diverse modes of recruitment
- Many IRESs require trans-acting factors to remodel the RNA for ribosome recruitment
What is APAF1?
Pro-apoptotic factor involved in stress response
How is APAF1 expression controlled by an IRES? (2)
- 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
