Translation Flashcards
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
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
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
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
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
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
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
8
Q
Class 2
A
- 3’OH nucleophilically attacks carbonyl group to directly form linkage between amino acid and tRNA
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
10
Q
Ribosome
A
- translates mRNA to protein
- enormous structures composed of protein and mRNA
- 2 subunits: large and small
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
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
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
14
Q
Process of Initiation in Prokaryotes
A
- small subunit begins translation
- 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 - only p site is available
- mRNA bound to the small subunit via the Shine Dalgarno sequence upstream of the start sequence/bound to the 16s ribosomal RNA
- positions AUG codon at peptidyl site: available to be bound by tRNA (complementary)
- 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)
- IF2 is a GTPase: upon correct binding GTP is hydrolyzed to release GDP
- hydrolysis requires the 50s subunit: binds to 30s unit to form full ribosome and all IF leave
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
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
- eIF3 blocks association between large and small subunits
- eIF1/1A bind to small subunit to block premature assembly/leaves only P site available
- initiator tRNA-Met bound to GTPase, eIF2 enters P site
- additional GTPase initiation factor eIF5B needed (preinitiation complex formed)
- mRNA ends checked by eIF4 before assembly with ribosome
- mRNA and eIF4 bind to ribosome and position 5’ end at beginning
- scanning of mRNA until AUG codon reached
- codon recognition stops movement and triggers 60s subunit binding - GTP hydrolysis = massive conformational change
20
Q
Prokaryotic Elongation
A
- A site empty for tRNA entry and check to see if anticodon can H bond to codon of mRNA
- tRNA selection random
- each tRNA chaperoned by EFTU bound to GTP (elongation factor)
- if there is H bonding, GTP hydrolysis triggered (GTPase activity)
- ejection of EFTU = conformational change in ribosome
- changes tRNA position in A site to fully occupy it
- 3’ end of initiator tRNA (P site) and next tRNA (A site)
- tRNAs now in close proximity
- 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
- catalysed by peptidyl transferase
- lone pair of N terminus (amino group) of 2nd amino acid attacks carbonly of first amino acid
- bond formation between amino acids
- carbonyl group reformed causing loss of tRNA bond in P site
- tRNA protonated by proton from ribose group and amine group deprotonated
- reformation of peptide bond and transfer of amino acid onto incoming tRNA in A site
- tRNA moves to half occupy P and A sites
- A site partly vacant : occupied by elongation factor G (GTP bound)
- conformational change - displacement of tRNA into P or E site
- mRNA moves along into free A site: translocation - 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
