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Molecular Biology > Translation > Flashcards

Translation Flashcards

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1
Q

Open Reading Frames

A
  • presence of start (AUG) and stop codons determines a codon sequence
  • any mRNA can have 3 possible ORF making different proteins but generally only one ORF is protein coding
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2
Q

Transfer RNA (tRNA)

A
  • adaptor to recognise triplet codon + link it to its amino acid
  • specific base pairing between triplet codon in mRNA and 3 bases in tRNA (anticodon)
  • amino acid covalently linked to the 3’ end
  • 1st base of the anticodon pairs with the 3rd base of the codon
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3
Q

Structure of tRNA

A
  • L shaped
  • anti codon at one end and amino acid at the other
  • directed by intramolecular base pairing and base stacking
  • amino acid arm, TC arm, D arm, anticodon arm
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4
Q

Wobble Base Pairing

A
  • interaction between 3rd anticodon base and 1st codon base is more flexible, allowing for other base pairings with similar H bonding
  • allows one tRNA to base pair with more than one codon (degeneracy of amino acid code)
  • wobble allowed because of a lack of helix, meaning there is less stringent steric criteria
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5
Q

Feature of tRNA

A
  • small
  • contain modified bases
  • phosphorylated 5’end (usually guanine)
  • activated amino acid attached to OH of invariant 3’ end: CCA
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6
Q

tRNA Charging

A
  • covalent linkage of amino acids to tRNA
  • not reliant on Watson Crick base pairing
  • specificity comes from exquisite ability of enzymes (amino acyl tRNA synthases) to recognise subtle differences in amino acid structure
  • 2 mechanisms: both require formation of AMP-amino acyl intermediate + nucleophilic attack of carbonyl group on the y-phosphoryl group of ATP
  • ability of enzymes to bind correct trna and amino acid ensures fidelity of translation
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7
Q

Class 1

A
  • use 2’OH to nucleophilically attack carbonyl of acyl-AMP
  • transfers electrons back onto the phosphate, leaving AMP
  • causes covalent linkage between amino acid and tRNA
  • 3’OH nucleophilically attacks amino acid (covalently linked)
  • protonation to reform 2’OH
  • transesterification to give amino acid linked to 3’ carbon
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8
Q

Class 2

A
  • 3’OH nucleophilically attacks carbonyl group to directly form linkage between amino acid and tRNA
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9
Q

tRNA Proofreading

A
  • ‘mischarge’ a tRNA by linking threonine tRNA with Serine
  • incubate with threonyl trna synthase
  • leads to rapid hydrolysis of mischarged tRNA to give free tRNA
  • this shows that if the wrong amino acid is incorporated, there is an editing function of the enzyme to hydrolyse the bond
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10
Q

Ribosome

A
  • translates mRNA to protein
  • enormous structures composed of protein and mRNA
  • 2 subunits: large and small
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11
Q

Ribosome structure

A
  • 3 trna binding sites: E, P, A
  • 16s ribosomal rna unit
  • eukaryotes: 40s and 60s units
  • prokaryotes: 30s and 50s units
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12
Q

Ribozymes

A
  • visualisation of the ribosome highlights the ability of RNA to form complex 3D structures used in chemical catalysis
  • a ribozyme is the protein and RNA together
  • the catalytic activity of RNA is extremely important in suggesting a biochemical basis for life
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13
Q

Initiation tRNA

A
  • requires specific tRNA for AUG start codon in ORF
  • all organisms have two tRNAs for the AUG methionine codon: one for initiation and one for internal methionine residues
  • bacteria have formyl-methionine on the initiator tRNA which is formed enzymatically after Met-tRNA synthetase links Met to tRNA-fMet
  • eukaryotes just use methionine for both
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14
Q

Process of Initiation in Prokaryotes

A
  1. small subunit begins translation
  2. E/A sites blocked by activity of protein factors (IF1 and 3)
    - IF1 blocks the A site to block tRNA entry
    - IF3 blocks the large subunit from binding prematurely
  3. only p site is available
  4. mRNA bound to the small subunit via the Shine Dalgarno sequence upstream of the start sequence/bound to the 16s ribosomal RNA
  5. positions AUG codon at peptidyl site: available to be bound by tRNA (complementary)
  6. initiation tRNA needs intiation factor 2 in order to bind the AUG codon in the p site (factor acts as a chaperone to the site)
  7. IF2 is a GTPase: upon correct binding GTP is hydrolyzed to release GDP
  8. hydrolysis requires the 50s subunit: binds to 30s unit to form full ribosome and all IF leave
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15
Q

Key Summary of Initation

A
  • IF1 and 3 bind to the small 30S subunit to prevent 50s assembly and tRNA entry into the A state
  • mRNA binds to the 30S unit using complementary base pairing with Shine Dalgarno sequence in the 16s rRNA of the 30S unit
  • fMet-tRNA brought to the P site with IF2-GTP
  • combines with the 50S unit after GTP hydrolysis and departure of initiation factors
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16
Q

Specificity of Interaction

A
  • correct incorporation of fMet-tRNA comes from mRNA, 16s rRNA interaction, AUG codon interaction with tRNA, and P site interaction with tRNA
17
Q

Shine - Dalgarno Sequence

A
  • found in mRNA
  • pairs with 16s rRNA of ribozyme
  • consensus sequence: AGGAGGU
18
Q

Eukaryotic Initiation

A
  • similar mechanism but more protein initiation factors and no SD sequence
  • positioning relies on initial interaction with both ends of the molecule (5’cap and 3’ polyadenyl tail)
  • assembles at the 5’ end as eukaryotic mRNAs are monocistronic (single ORF)
  • ribosome scans the mRNA in 5-3 direction until AUG codon is reached
19
Q

Steps of Eukaryotic Initiation

A
  1. eIF3 blocks association between large and small subunits
  2. eIF1/1A bind to small subunit to block premature assembly/leaves only P site available
  3. initiator tRNA-Met bound to GTPase, eIF2 enters P site
  4. additional GTPase initiation factor eIF5B needed (preinitiation complex formed)
  5. mRNA ends checked by eIF4 before assembly with ribosome
  6. mRNA and eIF4 bind to ribosome and position 5’ end at beginning
  7. scanning of mRNA until AUG codon reached
  8. codon recognition stops movement and triggers 60s subunit binding - GTP hydrolysis = massive conformational change
20
Q

Prokaryotic Elongation

A
  1. A site empty for tRNA entry and check to see if anticodon can H bond to codon of mRNA
  2. tRNA selection random
  3. each tRNA chaperoned by EFTU bound to GTP (elongation factor)
  4. if there is H bonding, GTP hydrolysis triggered (GTPase activity)
  5. ejection of EFTU = conformational change in ribosome
  6. changes tRNA position in A site to fully occupy it
  7. 3’ end of initiator tRNA (P site) and next tRNA (A site)
  8. tRNAs now in close proximity
  9. EFTU regenerated using GEF
    - GEF binds and replaces GDP for GTP, as the complex it forms with EFTU has a high affinity for GTP
    - charged EFTU now can bind tRNA
21
Q

mRNA checking in eukaryotes

A
  • mRNA looped around
  • eIF4 binds cap to position mRNA correctly
  • mRNA degradation occurs from the ends, so this formation protects them
  • checks mRNA integrity by checking the length of the polyadenyl tail
22
Q

Bond Formation

A
  1. catalysed by peptidyl transferase
  2. lone pair of N terminus (amino group) of 2nd amino acid attacks carbonly of first amino acid
  3. bond formation between amino acids
  4. carbonyl group reformed causing loss of tRNA bond in P site
  5. tRNA protonated by proton from ribose group and amine group deprotonated
  6. reformation of peptide bond and transfer of amino acid onto incoming tRNA in A site
  7. tRNA moves to half occupy P and A sites
  8. A site partly vacant : occupied by elongation factor G (GTP bound)
  9. conformational change - displacement of tRNA into P or E site
    - mRNA moves along into free A site: translocation
  10. GTP hydrolysis causing EFG ejection
23
Q

Peptidyl Transferase

A

23s ribosomal RNA in prokaryotes

catalytic site

24
Q

Molecular Mimicry by Proteins

A
  • EFTU resembles the structure of a tRNA

- similar in electron density and charge density

25
Q

Eukaryotic Elongation

A
  • same mechanism as prokayotic elongation

- different but analogous protein elongation factors

26
Q

Prokaryotic Termination

A
  • UAA, UGA, UAG are stop codons
  • no complementary tRNA: recognised by release factors RF1 and RF2
  • transfers peptide to water rather than amino acyl tRNA
  • hydrolysis of ester linkage to tRNA but without linking onto another tRNA (dissociation)
  • ribosome dissociation requires EF-G, RRF, IF-3 in prokaryotes or eRF in eukaryotes
27
Q

Inhibition of Protein Synthesis

A
  • naturally occuring toxins or antibiotics inhibit translation but eukaryote/prokaryote specificity implies structural differences between their ribosomes
    puromycin: binds A site causing premature termination
    tetracyclines: block A site / inefficient elongation
    chloramphenicol: blocks peptidyl transferase
    cycloheximide: binds eukaryotic peptidyl transferase
    streptomycin: causes codon misreading to form nonsense proteins
28
Q

Key Summary of Elongation

A
  • 3 elongation factors (EF-Tu, EF-Ts, EF-G)
  • EF-Tu GTP brings amino acyl tRNA into the A site
  • GTP hydrolyzed and EF-Tu GDP released if H bonding is complementary
  • peptidyl transferase activated
  • EF-Tu GTP regenerates using EFTs
  • peptide bond formation catalysed by 23S rRNA from nucleophilic attack by a-amino group of amino acid in the A site on carbonyl group of peptide in P site
  • translocation requires GTP hydrolysis + EF-G
  • moves one codon along
  • newly synthesized peptidyl-tRNA into the P site

Molecular Biology (11 decks)

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