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