Translation II Flashcards
1
Q
Ribosome Composition and Function
A
- Translate mRNA in 5’-3’ direction
- Can be seen in experiments: protein has same amino acids as translated going from N-terminal to C-terminal
2
Q
Similarities between eukaryotic and prokaryotic ribosomes
A
- Full complex: For translation, 2/3 rRNA folded into duplex regions, 1/3 proteins connect rRNA
- Small subunit: mRNA binding and codon/anticodon pairing
- Large subunit: Peptide bond formation
- Only rRNA at subunit interface
3
Q
Eukaryotic ribosome composition and function
A
- Made of 80S
- 40S small subunit with 30 proteins, 18S rRNA
- 60S large subunit with 40 proteins, made of 5S, 5.8S, and 28S rRNA
4
Q
Prokaryotic ribosome composition and function
A
- Made of 70S
- 30S small subunit with 21 proteins (S1-21), 16S rRNA
- 50S large subunit with 31 proteins (L1-31), made of 5S and 23S rRNA
5
Q
Prokaryotic translation initiation: sequence specificity
A
- First codon found 25 nucleotides downstream from transcript 5’ end
- To recognize codon, ribosomes bind to purine rich Shine Dalgarno sequence located 10 nucleotides upstream
- SD region recognized by 3’ end of 16S rRNA of small subunit
- Interaction positions AUG codon in P site
6
Q
Initiator tRNA
A
- AUG codon following SD sequence signals for formyl methionine residue carried by initiator tRNA
- Charge: initiator tRNA charged with methionine by Met-tRNA synthetase
- Modify: enzyme Met-tRNA formyl transferase adds formyl group to N terminal of tRNA bound Met. Mimics peptide bond to allow tRNA to bind to P site to begin translation. Regular Met and Met-tRNA cannot interact with formyl transferase
7
Q
Initiation factors
A
- IF1, 2, 3 are P loop NTPases that undergo conformational changes upon binding to NTPs
- IF1 and IF3 binding: 30S subunit binds both IF1 and IF3. IF1 binds to A site of 30S subunit ensuring initiator tRNA binds to P site. IF3 blocks premature binding of 50S subunit
- Binding of mRNA and IF2 to form 30S initiation complex: 16S rRNA will H-bond with mRNA transcript. IF2 binds to initiator tRNA and GTP. Forms 30S initiation complex
- Transition to 70S initiation complex: GTP hydrolysis leads to release of all initiation factors from complex. Promote binding of 50S subunit to form 70S initiation complex
8
Q
Prokaryotic Translation Elongation Mechanism
A
- Aminoacyl tRNA binding: Initiator tRNA binds to P site. Allows next aminoacyl tRNA to bind to A site. Anticodon region interact with small subunit, acceptor arm with large subunit
- Peptide bond formation: Takes place in P site of large subunit. Polypeptide transferred from tRNA in P site to A site. Acceptor arm undergoes positional shift, deacylated tRNA in P site move to E site, and polypeptide tRNA move from A to P site.
- Translocation: Ribosome shift by 3 nucleotide toward 3’ end of mRNA to position next codon in A site. Driven by hydrolysis of GTP and elongation factor G.
- Cycle: After elongation cycle, deacylated tRNA released from E site and return to initial state, having added one amino acid to polypeptide chain
9
Q
Aminoacyl tRNA binding
A
- Delivery of aminoacyl tRNA to A site facilitated by Ef-Tu bound by GTP. Protects ester linkage. When anticodon correctly pairs to mRNA, hydrolysis of GTP to GDP. Ef-Tu bound to GDP must be regenerated via guanine exchange factor Ef-Ts. Ef-Tu does not interact with the initiator tRNA -> brought in by IF-2 instead
10
Q
Peptide Bond Formation
A
- Amino group of amino acid in A site nucleophilically attacks carbonyl carbon of last amino acid in growing polypeptide chain
- Formation of tetrahedral intermediate -> undergoes bond rearrangement to form peptide bond
- After formation, acceptor arm of growing polypeptide tRNA while deacylated tRNA acceptor arm moves to E site
11
Q
Translocation
A
- GTP bound EF-G attaches to 50S subunit near A site.
- Hydrolyzes GTP to transfer tRNA one site over
- Translocates one codon toward 3’ end of mRNA transcript
- EF-G structure mimics Ef-Tu
12
Q
Cycle
A
- Ribosomes cycling through translation elongation process in groupings called polysomes
- Polysomes enhance transcriptional efficiency
13
Q
Mischarging tRNA molecules
A
- Base pairing between mRNA and tRNA dictate which amino acid added
- Specific amino acid does not play a role
- tRNACys correctly charged -> sulfur of Cys converted to hydrogen using solid catalyst Raney nickel to create Ala-tRNACys
- ## Combining Ala-tRNACys with template specific for cysteines creates polypeptide chain full of Alanines
14
Q
Wobble hypothesis
A
- First two nucleotides must form WC base pairing
- Third position has more freedom and can do Hoogsteen and WC base pairing
- eg. Guanine at position 3 can pair with Cytosine or Uracil
- RNA molecules can have inosine(I) that base pairs with most
- tRNA with I at first position can form Hoogsteen base pariing with A, U, C
- Flexibility of third position due to 16S rRNA only proofreading first two
- 16S rRNA has three nucleotides that H-bond with first and second position, as well as second and third position
- Expels tRNA if incorrect base pariring
15
Q
Prokaryotic transcription termination
A
- No aminoacyl tRNA complementary to STOP codons
- Recognized by release factor when at A site
- Binding of release factor: RF-1 or RF-2 binds to STOP codon assisted by GTP bound RF-3. RF-1 recognizes UAA and UAG and RF-2 recognizes UAA and UGA. RF-1 and 2 carry water molecule
- Peptide release: Water molecule used to hydrolyze ester bond between polypeptide chain and tRNA
- Complex dissociation: RF-3 hydrolyzes GTP to GDP leading to dissociation of release factors. Ribosomal release factor binds to ribosome and triggers release of entire translation complex using energy from another GTP
16
Q
Prokaryotic Translation Summary (w/ 9 amino acids)
A
- Charging tRNA using aminoacyl tRNA synthetases uses 2 ATP (one to form AMP and one to reform ADP): 2 x 9 = 18 ATP
- Translation Initiation required GTP hydrolysis by IF-2 to bring initiatior tRNA: 1 GTP
- GTP bound Ef-Tu brings in amino acids to ribosome via aminoacyl tRNA binding: 1x8 = 8 GTP
- Translocation occurs 8 times using EF-G and GTP hydrolysis : 1x8 - 8 GTP
- Translation termination requires 1 GTP from RF-3: 1 GTP
- Ribosome disassembly using RRF and GTP hydrolysis: 1 GTP
- 18 ATP and 19 GTP used to synthesize 9 amino acid protein
17
Q
Eukaryotic Translation Initiation
A
- Assembly of complexes: Begins with assembly of eIF4F heterotrimer complex, and 43S pre initiation complex. eIF4F consist of eIF4E that binds to 7 methyl G cap. 43S consist of small ribosomal subunit, initiator tRNA, and eIF2 bound to GTP to facilitate interaction between initiator tRNA and small ribosomal subunit. initiator Met not formylated in eukaryote.
- Complexes bind mRNA: Both complexes bind 5’ cap. eIF4F attach first, then 40S
- Circularization of mRNA: Undergoes circularization after binding to facilitate rebinding of ribosomes after translation. eIF4G component of eIF4F act as bridge between eIF4E and polyA binding protein at 3’ end
- Scanning of mRNA: Small ribosomal subunit scans for start codon. Eukaryotes do not have SD sequence and find first AUG sequence closest to 5’ end
- Recognition of initiation codon: When AUG encountered, interaction between mRNA and cognate tRNA trigger GTP hydrolysis of eIF2. Hydrolysis facilitate binding of 60S subunit and positioning initiator tRNA at P site to form 80S subunit. eIFs dissociate once fully assembled
18
Q
Puromycin
A
- Resembles adenine residue of tRNA acceptor arm
- Allows to fit in A site
- Forms amide linkage instead of ester linkage -> interfere with transcription
- Nitrogen atoms nucleophilically attacks C terminal of polypeptide chain forming peptide bond -> polypeptide bound to puromycin -> move to P site -> incoming amino acid cannot attack -> puromycin bound polypeptide dissociates and stops translation
- Affect eukaryotic and prokaryotic cells
19
Q
Streptomycin
A
- Prevents correct binding of initiator tRNA in prokaryotes due to its many amino groups
20
Q
Diphteria toxin
A
- Upon entering eukaryotic cell, toxin cleaved to A and B
- A catalyzes cleavage of NAD+ into ADP ribose and nicotinamide
- Attaches the ADP ribose to modified histine residue in EF2
- Inhibits EF2 ability to translocate
21
Q
Ricin
A
- N-glycosidase that inhibits protein synthesis in eukaryotes
- Heterodimer consist of A chain (catalytic activity) and B chain (cellular uptake)
- When ricin taken up, A chain released into cytoplasm and targets conserved adenine residue in 28S rRNA
- Cleaves N-glycosidic bond between adenine base and ribose sugar to prevent binding of elongation factors
