Lecture 8: Architecture of Translation Flashcards
What is translation?
- Joining of aminoacyl residues by ribosome to form polypeptide
- Primary sequence of the polypeptide is specified by the triplet codons in mRNA
- Encoded by triplets
- High energy cost to cell
Important that it only occurs when required
What are the components of the ribosome?
- Prokaryotic: 50S + 30 S = 70S
* 16S is smaller RNA prokaryotes
Eukaryotic: 60S + 40S = 80S
Are secondary structures of the ribosomes conserved?
- rRNAs of the E.coli were first sequenced in 1978
○ Secondary structure models proposed on bp alone- rRNAs from several hundred species hv been sequenced
○ All show the same defined structures - Conserved regions of rRNA are highly conserved [remain largely unchanged across different species]
○ Mutations in these conserved regions are often lethal - Variable regions can tolerate mutations
- Base-paired stems (α-form helix) is common
○ Compensating base changes occur in base-paired stems
○ Even though the specific base pairs may differ between species, changes occur in a way that maintains the overall structure and function of the ribosome.
- rRNAs from several hundred species hv been sequenced
What is essential in rRNA function?
- Stem loops
Demonstrated by a change in 1 bp in the loop results in another bp change (mutation) further up to maintain the structure & retain function
How are ribosomes structured?
- 3 binding sites for tRNA that span the 30S n 50S subunits
○ A: acceptor sit of codon-directed binding of incoming aa tRNA
○ P: peptidyl site, holds codon directed peptidyl tRNA
E: exit site, not associated w mRNA
How is the peptide bond formed b/w amino acid in ribosome?
- Peptidyl transferase reaction (nucleophilic addition)
- Catalyzes the formation of peptide bonds in amino acids
○ RNA driving the reaction - ribozyme activity - N3 accepts a proton from the amino group of the aminoacyl tRNA in the A site
○ Enhances negative charge of the amino group
○ Allows it to attack bond b/w peptide n tRNA in P site - N3 of A2486 H-bonds to oxyanion in the tetrahedral intermediate -> stabilizes n accelerates reaction
- 3’- OH of the tRNA in the P site accepts proton from A2486 -> completes reaction
- Catalyzes the formation of peptide bonds in amino acids
Are the proteins of the peptidyl transferase active site in the 50S subunit P site involved in catalysis?
- Nearest protein to the active site is 18.4 Angstrom from active site
Hence, too far to participate in catalysis
What is the poly-peptide exit tunnel in the 50S subunit?
- Exit tunnel for proteins to come out as its being formed
- Located in larger subunit of the ribosome
- Shape + size of exit tunnel, lots of hydrophobic residues -> allows protein to start folding, α-helical proteins come out alrdy
How are tRNAs named?
- According to the a.a. they’re charged with
- Eg. tRNA charged with alanine = tRNAala
What’s the difference between a charged and uncharged tRNA?
- Charged tRNA (aminoacyl-tRNA) has an a.a attached to it
- Uncharged one doesn’t
What’s the difference between an aminoacyl-tRNA and a pepNdyl-tRNA?
- Aminoacyl-tRNA= the tRNA molecule bound at A site
- Peptidyl-tRNA = the tRNA molecule bound at P site
What are isoaccepting tRNAs?
different tRNAs (often w different anticodon sequences) to become charged w the same amino acid
What are aminoacyl tRNA synthetases?
○ Enzymes which charge tRNAs
○ Shows specificity for the tRNAs they charge n the correct interaction is w cognate tRNAs
V rarely, non-cognate tRNA is amino acylated
Name the tRNA that contain a number of modified (unusual) nucleosides
- Dihydrouridine (DHU)
- Ribothymidine (T)
- Pseudouridine
- Inosine (I)
- Methylguanosine (mG)
Describe the cloverleaf model for tRNA
* D-loop
- D-loop
○ 8-12 unpaired bases
○ 2-3 dihydrouracil residues- Anticodon loop
○ 7 unpaired bases
§ Recognition b/w codon that’s on mRNA
§ Has to be opposite base for it to be able to bind to the right 3 bases to get correct amino acid insertion
○ 3 anticodon bases
○ Flanked on its 5’ side by U n on 3’ side by alkylated purine - Variable loop
- T loop
○ 7 unpaired bases
○ Involved in the binding to ribosome’s A site - Attached amino acid (Phe)
○ 3/ end always has CCA
○ A - amino acid attachment
Paired sections: STEMS b/w loops -> give structure, closely controlled
- Anticodon loop
Describe the teritary structure of yeast tRNA
- CCA-3’ is located ~70 A away from anitcodon
- DHU n TC loops form a L corner
- Most bases are stacked (major factor in stabilization)
- The 3 anticodon bases n the - CCA 3’ bases are unstacked
○ Allows interaction w codon base / aminoacyl tRNA synthetase - Many of the tertiary H-bonding interactions involve bases that are invariant in all known tRNAs
○ Supports belief that all tRNAs hv basically the same structure - H-bonds involve non-conventional AU n GC bp
What are shared and unique reactions of tRNAs?
- Shared
○ Interaction w elongation factor (except initiator tRNA)
○ Binding to the ribosome A site
○ CCA terminal addition
○ Invariant modifications to bases- Unique reactions of individual tRNAs
○ Amino acylation by synthetases
Codon-anticodon interaction
- Unique reactions of individual tRNAs
How do aminoacyl-tRNA synthetases charge tRNA?
- Synthase picks up correct AA
- Activation of the AA by picking up ATP (reaction catalyzed by aminoacyl-tRNA synthetase itself) -> pyrophosphate is released
- tRNA binds to synthetase
- Amino acid is covalently attached to tRNA
- AMP released
- When correct tRNA comes, interacts w AMP
Charged RNA released
What are cognate tRNAs?
- tRNAs recognized by amino-acyl synthetase enzymes
What are the features of individual tRNAs which are recognised by their cognate synthetase?
Identity elements, some lie in the anticodon some don’t
Describe the tRNA synthetase complex
- Editing site + activation site
- Editing site hydrolyses the bond with the AA -> prevents charging with wrong AA
What are the 2 stages of proofreading (double sieve) by aminoacyl-tRNA synthetases?
- Binding of cognate tRNA to synthetase -> Hydrolysis of the ester bond of an “incorrect” aminoacyl-AMP intermediate
- Hydrolysis of the ester bond of a “miss-matched” aminoacyl-tRNA
- Most aminoacyl-tRNA synthetases possess editing (hydrolytic) sites in addition to the acylation site
- Usually, the acylation site rejects an amino acid larger than the cognate aa [insufficient space
- The editing site hydrolyses aminoacyl-tRNAs which are smaller than the cognate aa.
If the aa is too small, won’t be held firmly
How are aminoacyl-tRNA synthetases edited?
- Flexible CCA arm of aminoacyl-tRNA can move the amino acid b/w activation n editing site
If the AA fits well into editing site -> AA is removed via hydrolysis
How does streptomycin inhibit protein synthesis?
- Highly basic trisaccharide
- Binds to the 16S rRNA of the 30S subunit of bacterial ribosome
- Interferes w binding of formyl methionyl-tRNA to ribosomes
RESULT: prevents correct initiation of protein synthesis
How does puromycin inhbit translation?
- Puromycin has a similar structure to aminoacyl-tRNA
○ Puromycin can spot itself into any vacant A site in the ribosome -> carry out peptide transferase action even tho it actually has no peptide to be attached to
○ Don’t get any further increase in length of the polypeptide
50% of ribosomes hv vacant A site so prone to puromycin -> stop translation
How does diptheria toxin inhibit translocation?
- Produced by pathogenic strains
- Extremely toxic toxin
- Acts catalytically on eukaryotic elongation of EF-2
- All EF-2s hv specific modification to histidine called diphtamide
○ Toxin transfers ADP ribose from NAD+ to the imidazole ring
○ Completely blocks translocation in the process of translation
How does diphtheria toxin enter cells and cause cell death?
- Diphtheria toxin accumulates on cell surface by interacting w sugars on cell surface
- Binds to growth receptor n pulled into the cell as a vesicle
- Gets cleaned by pyromin protease
- Released from the vesicle
- Disulfide bond that holds toxic n non-toxic part
- If cleaved, can interact w the cell -> cell death
How do ricin and sarcin inhibit protein synthesis in cells?
- Ricin n sarcin interact w the conserved stem loop structure of 28S rRNA that binds aminoacyl tRNA
○ RESULT: inhibits protein synthesis- Ricin: removes single adenine residue
- α-sarcin: single cleavage in the sugar-phosphate backbone
- Only need 1 ricin molecule in a cell bc it can snip a lot
How can ricin be used as a targeted toxin for cancer cells?
- Attaching toxin part of the protein to an antibody
- Recognition part of the antibody
- Targeted toxin that will go just to the cancer cell -> cancer cell will uptake -> kills cancer cells
