Protein Synthsis Steps Flashcards
Charged tRNAs
Aminoacylated
Enzyme for connecting tRNAs to matching amino acids
Aminoacyl-tRNA synthetase
Each enzyme is specific for only one amino acid
2 classes of synthetases
Different pathways for connecting amino acids
Each group helps about half of the amino acids
No clear common ancestor
Generic reaction catalyzed by aminoacyl synthesis
Amino acid + tRNA + ATP (Mg2+) —> aminoacyl-tRNA + AMP + PPi
Steps
Amino acid interacts with atp with carboxyl group oxygen as a nucleophile and and alpha phosphate of atp acceptor —> releases PPi
Aminoacyl-AMP enzyme bound intermediate is formed
Aminoacyl group transferred from intermediate to tRNA
Ester linkage betweeen tRNA and AA
Class I
Adenine terminal of tRNA has 2’ o attack carboxyl c —> releases amp —> flipped to 3’o
Class II
2’o attacks initially and relases amp
Product = aminoacyl-tRNA
Synthetase proof reading
Many but not all synthetases have proofreading abilities
Will have two active sites
One for both correct and incorrect to attach to
1 that only incorrect will attach to and be hydrolyzed
Additionally, synthetases hydrolyze ester linkages and this hydrolysis is accelerated when tRNA incorrectly charged
Error rate for protein synthesis
1 mistake per 10^4 correct
What is the second genetic code?
Synthetases’ need to identity amino acids and tRNAs
ID primarily by info in AA arm and anticodon arm
Class I and class II bind to opposite faces of substrate tRNA
For genetic code expansion:
Need: - new syntheses - new AA - new cognate tRNA
2 extra amino acids
- Selenoysine
= normal ser attached to tRNA but edited before ribosome - pyrrolysine
=unique tRNA and unique synthetase
Unnatural genetic code expansion
-geneses for tRNA and synthetase taken from certain organism
tRNA selected by anticodons don’t determine having synthetase help reaction so codon can be changed
Codon changed to least common stop anticodon = CUA
Need to make sure endogenous synthetase doesn’t work on tRNA so put in plasmid along with another plasmid that has Barnard gene
If tRNA is used to translate Barnard gene functionally rather than as a series of stop codons, the. Endogenous synthetase is working = dead cell bc Barnard deadly
= alive good for more testing = negative selection
Need to make sure that modified synthetases are specific only modified tRNAs
1 plasmid has both synthetase genes and B lactase gene
Other has tRNA gene edited with lots of UAG
If lac expressed then can live on ampicillin
Which is proof of tRNA and synthetase interaction
If it could interact with old tRN for UAG, b lac could not be expressed
= positive selection
All organisms have one codon but two tRNAs for what codon?
AUG (Met)
One for initiation
One for met within polypeptide
Bacteria name different for two different aug codons
fMet and Met
Bacterial initiation mechanism
2 steps
Methionine + tRNA + ATP —> met-tRNA^fMet + AMP + PPi
N-formeletetrahydrofolate + met-tRNA^fMet —> fmet-tRNA^fMet + tetrahydrofolate
Via transformylase
This can now no longer be added to internal peptide due to N formyl physical blockage
Eukaryotic cells initiate with
Met reside but two different tRNAs depending on initiation or peptide bonding
BUT mitochondrion and chloroplasts do use fMet
IF1
Prevents premature binding to A site
IF2
Facilitates fMet-tRNA^fMet binding to 30S subunit
IF3
Prevents premature 30s and 50s subunit interaction
ElF1A (eukaryotic)
Prevents premature A site binding
IF1 homolog
ElF1 (eukaryotic)
Binds to 40S E site
Helps tRNA-elF2-GTP complex bind to 40S
elF2 (eukaryotic)
GTPase
Helps 40S + met-tRNA^Met binding
elF2B (eukaryotic) + elF3 (eukaryotic)
1st to bind to 40S subunit
Help with all subsequent steps
elF4E (eukaryotic)
5’ cap of mRNA binding
Mediates association with 43 S complex
elF4A (eukaryotic)
ATPase
RNA helicase
Removes secondary structure of mRNA for 40S binding
elF4G (eukaryotic)
Binds to polyA binding protein to help with mRNA circularization
Linker protein
Works either elF4E and elF3 to form first link between 40s subunit and mRNA
elF4F (eukaryotic)
Complex with elF4E elF4G, elF4A
elF4B (eukaryotic)
Binds to mRNA
Helps scan for AUG
elF5 (eukaryotic)
Promotes initiation factors dissociation from 40S —> prepping of 80S
elF5b (eukaryotic)
GTPase that promotes dissociation of initiation factors prior to final ribosome assembly
Homolog to IF2
Bacterial initiation
30S + if1 + if3
30s complex + mRNA
=alignment via Shane dalgarno sequence
=8-13 bps A + G of 5’ mRNA end
Align with c + u in 16S rRNA
Initiating AUG = at P site to which fMet - tRNA ^fMet binds
This complex bindss with 50S subunit
IF2 hydrolzyed and uses up GTP
Initiation factors released
=70S initiation complex
Matching = codon anticodon interaction, tRNA fitting into p site, Shane dalgarno for mRNA alignment
3 types of elongation factors
EF-Tu
EF-T
EF-G
EF-Tu
EF-Tu-GTP complex helps aminoacylated tRNAs bind to A site of 70S initiation complex
Binding releases EF-Tu- GDP and PPi
EF-T comes to bind to EF-Tu to release GDP
recycled when EF-Tu replaces EF-T with GTP
Initial peptide bond formation
Transfer of N-formylmethionine group connected to fMet tRNA in p site to amino group of amino acid at A site
N of AA is nucleophile to carboxylic Carbon
Produces h2o
Initial peptide bond formation
Transfer of N-formylmethionine group connected to fMet tRNA in p site to amino group of amino acid at A site
N of AA is nucleophile to carboxylic Carbon
Produces h2o
EFP
Binds E and Pnsites for stability esp during double proline addition bc side chains are tricky
If efp lacking - lots of ribosomal stalling = health issues
Eukaryotic homolog: elF5A
TmRNA and SmpB
Transfer messenger RNA and small protein B
Part of trans-translocation system that saves translation when mRNA stops before stop codon
TmRNA binds to A site until stop codon within tmRNA found and ribosome is recycled
Peptidyl transferase
Catalyzes peptide bond formation
Within 23S rRNA
Aligns with tRNA and a and p sites
EF-G
AKA translocase
Helps shift over mRNa
Needs GTP energy
Allosteric change during translocation
New AA in A site leads to structure change that forces uncharged tRNA from E site
Proofreading and EF-Tu
Time for association and release of GDP = time to check codon and anticodon fidelity
(Cannot double check correct AA is on tRNA)
Termination codons
UAA
UAG
UGA
RF1
Recognizes UAG and UAA
RF2
Recognizes UGA and UAA
RF1 and RF2
Have polypeptide transfer to h2o molecule
RF3
Thought to belong ribosomal subunit release
Ribosomal recycling
EF-G + RRF
GTP is hydrolyzed and they are released
Replaced by IF3
= tRNA dissociation
Polysome
Large cluster of ribosomes
In bacterial
Same mRNA that is still being transcribed
