Lecture 8: Architecture of Translation

Question 1

What is translation?

Answer 1

• 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

Question 2

What are the components of the ribosome?

Answer 2

• Prokaryotic: 50S + 30 S = 70S
  * 16S is smaller RNA prokaryotes
  Eukaryotic: 60S + 40S = 80S

Question 3

Are secondary structures of the ribosomes conserved?

Answer 3

• 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.

Question 4

What is essential in rRNA function?

Answer 4

• 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

Question 5

How are ribosomes structured?

Answer 5

• 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

Question 6

How is the peptide bond formed b/w amino acid in ribosome?

Answer 6

• 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

Question 7

Are the proteins of the peptidyl transferase active site in the 50S subunit P site involved in catalysis?

Answer 7

• Nearest protein to the active site is 18.4 Angstrom from active site
  Hence, too far to participate in catalysis

Question 8

What is the poly-peptide exit tunnel in the 50S subunit?

Answer 8

• 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

Question 9

How are tRNAs named?

Answer 9

• According to the a.a. they’re charged with
• Eg. tRNA charged with alanine = tRNAala

Question 10

What’s the difference between a charged and uncharged tRNA?

Answer 10

• Charged tRNA (aminoacyl-tRNA) has an a.a attached to it
• Uncharged one doesn’t

Question 11

What’s the difference between an aminoacyl-tRNA and a pepNdyl-tRNA?

Answer 11

• Aminoacyl-tRNA= the tRNA molecule bound at A site
• Peptidyl-tRNA = the tRNA molecule bound at P site

Question 12

What are isoaccepting tRNAs?

Answer 12

different tRNAs (often w different anticodon sequences) to become charged w the same amino acid

Question 13

What are aminoacyl tRNA synthetases?

Answer 13

○ 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

Question 14

Name the tRNA that contain a number of modified (unusual) nucleosides

Answer 14

• Dihydrouridine (DHU)
• Ribothymidine (T)
• Pseudouridine
• Inosine (I)
• Methylguanosine (mG)

Question 15

Describe the cloverleaf model for tRNA
* D-loop

Answer 15

• 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

Question 16

Describe the teritary structure of yeast tRNA

Answer 16

• 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

Question 17

What are shared and unique reactions of tRNAs?

Answer 17

• 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

Question 18

How do aminoacyl-tRNA synthetases charge tRNA?

Answer 18

• 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

Question 19

What are cognate tRNAs?

Answer 19

• tRNAs recognized by amino-acyl synthetase enzymes

Question 20

What are the features of individual tRNAs which are recognised by their cognate synthetase?

Answer 20

Identity elements, some lie in the anticodon some don’t

Question 21

Describe the tRNA synthetase complex

Answer 21

• Editing site + activation site
• Editing site hydrolyses the bond with the AA -> prevents charging with wrong AA

Question 22

What are the 2 stages of proofreading (double sieve) by aminoacyl-tRNA synthetases?

Answer 22

• 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

Question 23

How are aminoacyl-tRNA synthetases edited?

Answer 23

• 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

Question 24

How does streptomycin inhibit protein synthesis?

Answer 24

• 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

Question 25

How does puromycin inhbit translation?

Answer 25

• 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

Question 26

How does diptheria toxin inhibit translocation?

Answer 26

• 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

Question 27

How does diphtheria toxin enter cells and cause cell death?

Answer 27

• 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

Question 28

How do ricin and sarcin inhibit protein synthesis in cells?

Answer 28

• 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

Question 29

How can ricin be used as a targeted toxin for cancer cells?

Answer 29

• 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