Chapter 22 and 23 - Translation Flashcards
steps of translation
initiation
elongation
termination
initiation
The steps of translation up to entrance of the first aminoacyl-tRNA into the A site
elongation
Repeated rounds of polypeptide chain extension
Addition of one amino acid at a time to the growing polypeptide chain
termination
A separate reaction that ends translation by stopping the addition of subunits and stimulating disassembly of the apparatus
a site
aminoacyl tRNA
p site
peptidyl-tRNA
E site
deacetylated tRNA exit
ribozymes
ribosome with catalytic site
all of them!
tRNA
3’ terminus has the sequence 5’-CCA-3’ that serves as the amino acid acceptor stem
Presence of unusual bases in their structure
All created from standard ribonucleotides post-transcriptionally
tRNA structure
Have a characteristic and conserved pattern of single and double stranded regions
Amino acid acceptor arm
ΨU (Pseudouridine) loop
Dihydrouridine (D) loop
Anticodon loop
Variable loop
Often represented as a cloverleaf structure
Actual structure is inverted L-shape
aminoacyl-tRNA synthetase
are the family of enzymes that load tRNAs with the correct amino acid
aminoacyl-tRNA synthetase steps
- An amino acid reacts with ATP to form an aminoacyl adenylate intermediate
Energy of hydrolysis is trapped in the mixed anhydride linkage of the adenylate
Pyrophosphate is released - The 2’-OH or 3’-OH of the terminal 3’ nucleotide in the tRNA attacks the carbonyl carbon of the adenylate
- An aminoacyl-tRNA and AMP is formed
how do the synthetases detect tRNA differences
All tRNAs share the same general tertiary structure, but differ at nucleotide positions of the four arms
Changes in the nucleotide sequences
Subtle differences between the shape of the L-shaped arms
tRNA synthetases discriminate between tRNAs using both direct (nucleotide differences) and indirect (phosphodiester) methods
Most common discriminators are in the anticodon loop and amino acid acceptor arm
how do synthetases detect amino acid differences
Primary discriminator is shape of different amino acids
But amino acids are very small, and some are very similar in structure
Those that are similar in structure have different binding efficiencies and free energies
class I tRNA synthetases
Primarily monomeric
Aminoacylate tRNA at 2’-OH
Bind tRNA in the minor groove of the amino acid acceptor stem and require hairpin formation
Reaction rate is limited by rate of aminoacyl-tRNA release
class II tRNA synthetases
Primarily dimeric
Aminoacylate tRNA at 3’-OH
Bind tRNA in the major groove of the amino acid acceptor stem
Reaction rate is limited by previous chemical steps or active site rearrangement
synthetase kinetic proofreading when correct
tRNAs that match the specific nucleotide sequence combination for the synthetase
Properly align their amino acid acceptor stem with the ATP and amino acid in the active site
Quickly trigger aminoacylation reaction
synthetase kinetic proofreading when incorrect
Misalignment of acceptor stem in active site
Will not quickly trigger aminoacylation reaction
Dissociates much faster than it can react
isoleucyl synthetase chemical proofreading
Isoleucyl-tRNA synthetase cannot effectively distinguish isoleucine from valine using shape of amino acid binding site
Unable to prevent significant levels of valine-tRNAIle synthesis without proofreading
Nine different tRNA synthetases are able to proofread and correct errors once incorrect amino acid has bound to enzyme
Analogous to the 3’to5’ exonuclease proofreading function of DNA polymerases
two forms of chemical proofreading
pre-transfer editing
post transfer editing
pre-transfer editing
Incorrect aminoacyl-AMP is hydrolyzed after tRNA binding but before charging has occurred
post-transfer editing
Amino acid is hydrolyzed from aminoacyl-tRNA after tRNA charging
Uses an editing active site in the synthetase enzyme that is separate from the synthetic/loading active site
post transfer editing sieve analogy
The post-transfer editing pathway can be thought of as an integrated double-sieve
Based on relative sizes of the synthetic and editing sites
The synthetic site is larger than the editing site
The first sieve is the synthetic site
Amino acids larger than correct amino acid will be excluded from the synthetic site
Loading will not occur
The second sieve is the editing site
Amino acids smaller than the correct amino acid will fit into the synthetic site and the editing site
The incorrect amino acid will then be hydrolyzed and removed in the editing site
post transfer editing mechanism
Amino acids larger than correct amino acid will be excluded from the synthetic site
Loading will not occur
Amino acids smaller than the correct amino acid will fit into the synthetic site and the editing site
The incorrect amino acid will then be hydrolyzed and removed in the editing site
The correct amino acid can fit into the synthetic site, but not the editing site
Will be correctly charged and retained in an aminoacyl-tRNA
in bacteria, where does initiation occur
start codon and shine-delgarno sequence
bacterial steps of making ribosome
- 30S subunit binds to mRNA first, aided by initiation factors
- A 30S subunit carrying several initiation factors binds to an initiation site on mRNA to form an initiation complex
- All initiation factors are then released and the 50S subunit joins to form the full ribosomal structure
IF 3
stabilizes the free 30S subunit and must eventually be released to allow the 50S subunit to join the 30S-mRNA complex
also helps the 30S subunit bind to the initiation sites on the mRNA
IF 2
aids binding of the initiator tRNA to the complex
IF 1
binds to the 30S subunit at the A site and prevents aminoacyl-tRNAs from binding prematurely
formyl methionine
tRNAfMet
The aminoacyl-tRNA that initiates bacterial polypeptide translation
The amino group of the methionine is formylated
The methionine is formylated … it has been loaded onto the initiator tRNA
after
The initiator tRNA has unique … features that distinguish it from all other tRNAs
structural
The only initiation complex component that can bind the mRNA is the
small subunit
The initiator tRNA is the only tRNA able to bind to the … contained within the 30S subunit
partial P site
The small ribosomal subunit in eukaryotes recognizes the … of the mRNA and moves to the initiation site
5’ cap
Scanning model of eukaryotic initiation
Small subunit binds to the 5’ cap and begins to move 5’3’ down the mRNA
As it moves, the small subunit can melt some secondary structures of the mRNA
The small subunit stops when it recognizes the start codon and flanking sequences at -4 and +1
Kozak sequence
Weak Kozak consensus can lead to “leaky scanning”
Uniqueness of eukaryotic initiator tRNA is contained only in its structure
Phosphorylation of 2’-OH on nucleotide 64
Entry of an aminoacyl-tRNA into the A site is mediated by
EF-Tu
bacterial elongation steps
- EF-Tu-GTP binds an aminoacyl-tRNA and escorts it to the ribosome
- The anticodon end of the ternary complex moves into the A site of the 30S subunit and base pairs with the codon
- The EF-Tu-GTP end of the ternary complex binds to the factor binding center of the large subunit
- The factor binding center simulates EF-Tu-GTP hydrolysis
- The aminoacyl end of the aminoacyl-tRNA moved to face the P site tRNA
- EF-Tu-GDP is released
- EF-Ts mediates the regeneration of EF-Tu-GTP
- Peptidyl transferase
Creation of a peptide bond between the amino acid on the A site tRNA and the polypeptide on the P site tRNA
Attack of the amino group on aminoacyl-tRNA on the acyl linkage of peptidyl-tRNA - Upon transfer of the polypeptide to the A site tRNA, the tRNAs move into their hybrid state
- EF-G-GTP binds to the ribosome and stabilizes the hybrid state
- Binding of EF-G-GTP to the factor binding center stimulates GTP hydrolysis
- EF-G-GDP “unlocks” the ribosome by opening the gates between the E, P, and A codon-anticodon binding sites
- EF-G-GDP also binds to the A site in the codon-anticodon region
- A site anticodon is pushed to the P site
- P site anticodon is pushed to the E site
- The small subunit rotates and the ribosome now has a reduced affinity for EF-G-GDP
- EF-G-GDP dissociates
- EF-G has a lower affinity for GDP than GTP, so it will release GDP and rapidly bind a new GTP
- The system is now ready for another round of elongation
in bacteria, When there is correct base-pairing between first two positions of the codon and anticodon
16S rRNA forms nonspecific hydrogen bonds with the minor groove of the A site tRNA
Correctly base paired tRNAs dissociate from the A site very slowly, but incorrectly base paired tRNAs dissociate quickly
EF-Tu as a proofreader
EF-Tu-GTP only interacts with the factor binding center if the aminoacyl-tRNA fully enters the A site
Only occurs if there is correct base pairing between the codon and anticodon
An incorrect aminoacyl-tRNA will dissociate before EF-Tu-GTP interacts with the factor-binding center and promotes GTP hydrolysis
Hydrolysis to EF-Tu-GDP would commit the incorrect amino acid to incorporation into the growing polypeptide
The aminoacyl-tRNA initially binds to the A site with the amino acid oriented away from the P site
When EF-Tu-GTP is hydrolyzed to EF-Tu-GDP
The A site tRNA rotates towards the P site and the peptidyl transferase center
Accommodation
Correctly base paired tRNA will handle the rotational strain
Incorrectly base paired tRNA will be unable to handle the rotational strain
Base pairs will break
tRNA will dissociate from A site
entropic catalysis
Amino group of amino acid on aminoacyl-tRNA placed in close proximity to the carbonyl of the last amino acid added to the peptidyl-tRNA
proximity stimulated
substrate-assisted catalysis
The rRNA and P site tRNA also directly participate in the reaction in an enzymatic fashion
2’-OH of A2451 in 23S rRNA
2’-OH of P site tRNA and proton shuttle
hybrid state
The anticodons of the tRNAs remain in their pre-peptidyl transfer positions
The 3’ end of the A site tRNA is now bound to the polypeptide and prefers to bind in the P site
The 3’ end of the P site tRNA has now been deacylated and prefers to bind in the E site
Termination steps
- The termination codons are recognized by class 1 protein release factors
- RF3-GDP binds to the ribosome in the presence of bound class 1 factor
- Release of the polypeptide by the class 1 factor stimulates a change in the conformation of both the ribosome and the class 1 factor
- This stimulates RF3 to exchange bound GDP for GTP
- RF3-GTP will facilitate a transition of the ribosome into the hybrid state
- The class 1 factor dissociates
- RF3-GTP now associates with the factor binding center and be hydrolyzed to RF3-GDP
- RF3-GDP is released
- RRF binds to the empty A site and recruits EF-G-GTP to the ribosome
- EF-G-GTP is hydrolyzed to EF-G-GDP
- Unloaded tRNAs are released from the E and P sites
- EF-G-GDP, RRF, and the mRNA are released from the ribosome
- IF3 binds to the small ribosomal subunit, resulting in the dissociation of the subunits
RF1
recognizes UAG and UAA
RF2
recognizes UGA and UAA
eRF1
the only class I termination release factor in eukaryotes
RF3
class 2
required GTP
Class 2 release factors help … the class 1 factor from the ribosome
release
All three release factors…
have structures similar to that of EF-Tu-tRNA and EF-G
All bind GTP
All bind to the factor binding center or the A site
class 1 release factors
RF1 and RF2
contain a three amino acid peptide anticodon that recognizes and interacts with the stop codons
contain a GGQ motif that is placed in the vicinity of the peptidyl transferase center in the large subunit
The class 1 factors have structure where the peptide anticodon is on one end of the molecule and the GGQ is on the other
Similar to the anticodon and CCA stem in a tRNA
The release factors catalyze use of a water molecule as an acceptor of the peptidyl transferase reaction
Rather than an aminoacyl-tRNA
RRF
Dissociation of the remaining translation components requires the ribosome recycling factor
23S rRNA
Interacts with 3’-CCA terminus of P site tRNAs in peptidyl transferase center
Removing almost all proteins from the 50S subunit results in a 23S rRNA complex (with protein fragments) that still retains peptidyl transferase activity
23S rRNA alone has a low level of peptidyl transferase activity
Archaeal large subunit has only 23S rRNA in the peptidyl transferase center
Directly or indirectly involved in removing proton from amino group of peptidyl-tRNA
Proton shuttle?
