Translation Initiation Flashcards
What is translation?
The conversion of mRNA into a growing peptide chain of amino acids
What are anti-codons?
o Each codon is associated with an anti-codon
o Anti-codon comes from tRNA that carries an amino acid
o Also read 5’ to 3’
o A codon and its anti-codon are complementary
How do peptides grow? (Include the direction)
• Peptides grow from N-terminal to C-terminal
o The incoming amino acid will join its N-terminus on the last amino acid’s C-terminus
o The first amino acid has a free N-terminus
In which direction does the ribosome move down the mRNA?
5’ to 3’
What are ribosomes? (What are they made of?)
All ribosomes are large ribonucleoprotein complexes (protein that contains RNA)
Compare bacterial vs. mammalian ribosomes
o 70s vs. 80s
o Size: mammalian is larger
o Amount of RNA: bacteria have more
o Number of proteins: mammalian have more
o Large and small subunits (both larger in mammals)
What are the roles of rRNA?
1) structural role
2) catalytic activity
What are the roles of ribosomal proteins
1) structural role
o Provide ribosomal site for translation
o Form 3d active sites to facilitate translation
o Provide proximity
o Provide scaffolding by binding to rRNA or other proteins
2) catalytic roles
What are the major ribosomal binding sites?
• A site: aminoacyl site
o Binds incoming tRNA (codon/anti-codon recognition)
• P site: peptidyl site
o Site of addition of amino acid to growing chain
• E site: exit site (only in bacterial ribosomes)
o Deacylated (empty) tRNA exits the ribosome after the ribosome has moved forward 1 codon
What are the stages of translation?
1) Initiation: all reactions prior to the first peptide bond
2) Elongation: all reactions between the 1st and the last peptide bond
3) Termination: all reactions following the last peptide bond
I. Release of completed protein
II. Disassembly of the intact ribosome
What are the steps of initiation?
- Small subunit binds to mRNA
- Binding of first tRNA
- Completion of ribosome: bringing of large subunit
What are the factors involved in bacterial translation initiation?
o IF-1
Binds close to partial A site (the A site is completed when the ribosome is completed)
Only as part of the initiation complex
o IF-2
1. Binds the initial AA-tRNA and controls entry into partial P site
2. GTPas activity: GTP hydrolysis
• Provides energy for the LSU to SSU binding
• Results in conformational change, allowing release of Ifs
o IF-3
Binds free cytosolic SSU, maintaining the dissociated state of ribosome in the cytosol
Facilitates SSU binding to mRNA RBS (ribosome binding site)
Confirms accuracy of initator AA-tRNA
What is the RBS?
Ribosomal binding site.
Based on 2 highly conserved sequences
o Shine-Dalgarno sequence: AGGAGG
o Translation initiation codon: AUG
Describe the 1st step of bacterial translation initiation.
- Base pairing ensures that the SSU docks at the right place (i.e. on the RBS)
- IF-1 blocks potential entry of any tRNA
- IF-3 also binds the SSU
What is the initial bacterial tRNA?
tRNAMetF
What are the types of methionine that exist in bacterial cells and what distinguishes them?
1. tRNAMetF Only in initiation Binds IF-2 It has an unusual tertiary structure f-met contains a formyl group (it is formylated) fMet-tRNAF is the initation AA-tRNA 2. tRNAMetM Only in elongation tRNA with a conserved structure It binds the intact A site only
Describes the 3rd step of bacterial initiation.
- Once the joining is completed, translation shifts from initiation to elongation
- GTP hydrolysis (GTPGDP) releases energy that induces conformational change that releases IF-2
- IF-1 and IF-3 are released
- The incoming AA-tRNA can bind
What are the types of methionine that exist in eukaryotic cells and what distinguishes them?
• Initiator AA-tRNA is Met-tRNAI
o There are 2 different eukaryotic tRNAMet
tRNAMetI : used for initiation
tRNAMet¬M : used for elongation
• SSU binds to the 5’ G-cap of mRNA (which is away from the AUG initiation codon)
• More initiation factors involved than bacterial initiation
o eIF (analgous to bacterial IF)
o Binding order of factors is different
Where does the initiation complex form?
At the 5’ G-cap (away from the AUG initiation codon)
What is the coorrect consesus sequence for eukaryotic initiation?
1 purine, 3 nucleotide upstream of AUG
5’G ……..NNN(A⁄G)NNAUGG
What is the 43S complex? What elements does it comprise? Where does it form?
43S pre-initiation complex
o Factors: eIF2, eIF3, eIF1, eIF1A
o Met-tRNAI
Forms on the eukaryotic SSU (40S).
What are the roles of eIF2?
o Binds and docks the initiator Met-tRNAI
o Has GTPase activity
What are the roles of eIF3?
o Binds and maintains the dissociated state of SSU
What are the roles of eIF1 and eIF1A?
o Enhance the dissociation function of eIF3
What is the cap binding complex? What does it comprise? Where does it form?
It forms on the mRNA • eIF4E • eIF4A • eIF4B • eIF4G
What are the roles of eIF4E?
o Cap binding subunit
What are the roles of eIF4A?
o Helicase activity
o Unwinds the secondary structure at the 5’end of mRNA
What are the roles of eIF4B?
o Stimulates the helicase activity
What are the roles of eIF4G?
o Scaffolding subunit
o mRNA needs to be circularized prior to translation
o Connects the 5’G cap and 3’PABP/Poly A tail (circularizing mRNA)
What is the 48S scanning complex? What does it comprise? Where is it formed?
• 43S complex binds to the cap-binding complex on the 5’ end of mRNA Factors • eIF1 and eIF1A • eIF5 • eIF5B
What are the roles of eIF1 and eIF1A?
o Facilitate movement from 5’ to 3’
What are the roles of eIF5?
o Facilitates the eIF2 GTPase activity, which then releases eIF2 and eIF3
What are the roles of eIF5B?
o chaperones LSU into correct position
o Formation of intact ribosome
