Translation Flashcards
Translation
mRNA directed polypeptide synthesis
Ribosome
Structure made of proteins (~50) and rRNA (3-5) which catalyzes peptide bond formation
–> The actual place/machine where translation occurs
tRNA
Transfer RNA –> The “translator”
–> “Adapter” molecules which LINK info in mRNA codons with specific amino acids
tRNA Synthetases
20 of them
–> Enzymes that recognize specific tRNAs and amino acids and causes the attachment of AAs to tRNAs
Molecular components needed for translation: (4)
1) Ribosome
2) tRNA
3) tRNA synthetases
4) At least 9 soluble accessory proteins
What is the role of accessory proteins in translation?
Have functions in regulating steps of translation (initiation, elongation, and termination)
Translation uses…
But cells are willing to do it because…
1) more energy than any other biosynthetic pathway
2) It is so fundamental to life
tRNA structure
CLOVERLEAF Structure
–> This is a 3D structure that is highly structured
How long are tRNAs roughly?
typically ~80 nucleotides long
How does intra-molecular base pairing impact tRNA structure?
Causes the molecule to fold in on itself producing its characteristic loops and stems
–> Creates a 3D structure
What causes the specific structure of tRNA?
INTRA-molecular base pairing (the RNA base pairs with ITSELF)
“free”/exposed sections of the tRNA molecule:
1) Amino acid attachment site (3’ End of the tRNA)
2) Anti-codon region
What does “free” or “exposed”-regions mean in terms of tRNA?
Sections of tRNA not involved in intra-molecular base pairing therefore leaving the nucleotides “free” and exposed (able to interact with other things)
Protruding 3’ End of tRNA sequence =
CCA (5’ –> 3’)
What is the amino acid attachment site?
Where an amino acid is covalently attached to a tRNA
–> This site is located on the free 3’ end of a tRNA molecule
Anti-Codon
A particular nucleotide triplet on a tRNA that base pairs to a specific mRNA codon
Anticodons are written…
Because…
3’ to 5’
–> To more easily show the anti-parallel pairing of codon and anti-codon
Functions of tRNA structure (3)
1) “fit” in the ribosome
2) Interaction with correct codon
3) Correct amino acid attachment
tRNAs are NOT _____________
–> They differ in:
NOT identical
Differ in:
1) Each has a specific covalently attached amino acid
2) Each has a unique anticodon
Each tRNA is continuously….
recycled
–> a tRNA gained a designated amino acid in the cytosol where it then deposits the amino acid at the ribosome
–> Once deposited, it returns to the cytosol to pick up another amino acid (recycling itself)
TWO recognition events must occur for proper translation (as a whole):
1) tRNA anticodon must pair to correct mRNA codon
2) tRNA must carry the correct amino acid
Pairing of tRNA and amino acid is mediated by:
An enzyme –> Aminoacyl-tRNA synthetase
Aminoacyl-tRNA synthetase
A family of enzymes which catalyze the attachment of an amino acid to the 3’ end of the tRNA
Active site of aminoacyl-tRNA synthetase
Active site of these enzymes fit only a specific combo of AA and tRNA
recognition of specific tRNA by tRNA-synthetase
Occurs through recognition of specific residues in the anti-codon and amino-acid accepting arm/site
– >Allows correct tRNA to be recognized by the enzyme
If the wrong amino acid is connected to a tRNA…
Translation WILL NOT STOP
–> Translation will continue on and just produce an incorrect protein
Why is correct recognition of tRNA by tRNA synthetases especially important?
Because there is NO proofreading mechanisms for attaching AAs to tRNAs
Raney Nickel Experiment proved…
tRNA is the adapter molecule between protein and mRNA
Raney Nickel Experiment: Methodology
1) Purified a tRNA with cysteine anti-codon and cysteine AA attached (tRNA-cys)
2) Incubated tRNA-cys with RANEY NICKEL
3) Raney nickel converted the AA on tRNA-cys to ALANINE
–> Produced tRNA with cysteine anti-codon BUT ALANINE AA (anticodon and AA didn’t match): “ala-tRNA-cys”
4) Inserted this ala-tRNA-cys into an in-vitro hemoglobin synthesis system
5) Analyzed hemoglobin product
Raney Nickel Experiment: Results
Hemoglobin protein produced had ALANINE at every position normally occupied by cysteine
Raney Nickel Experiment: Conclusion
The amino acid attached to the tRNA is passively brought to the mRNA and becomes part of the polypeptide dictated by
CODON-ANTICODON INTERACTIONS
–> The actual amino acid on the tRNA has no effect on the recognition event between the codon and anti-codon
Ribosome Structure
Has 2 subunits: One large and one small
Ribosome subunits come together to form ____________ ONLY when…
to form a functional ribosome complex
ONLY WHEN attached to an mRNA molecule
Ribosomes of prokaryotes and eukaryotes are largely ___________ but have slight _____________
1) Largely similar
BUT
2) Have SLIGHT differences
Implications of ribosomal differences between prokaryotes and eukaryotes
Major medical implications –> Specifically with antibiotics
Role of ribosomes and ribosomal differences in antibiotics
Antibiotics must have SELECTIVE KILLING of bacteria
–> Must be able to kill bacteria but not us
–> Many antibiotics inhibit bacterial protein synthesis by targeting ribosomal components that differ from eukaryotes
rRNA is a…
ribozyme
What component of the ribosome catalyzes peptide bond formation?
rRNA
(NOT protein)
Translation Process Steps
1) Initiation
2) Elongation
3) Termination
Translation requires energy input from…
GTP hydrolysis
Initiation
Phase that brings together:
1) an mRNA
2) tRNA bearing first AA of polypeptide (initiator)
3) both ribosomal units
…to create the translation initiation complex (essentially the set up for protein synthesis to begin)
Initiation Process (steps)
1) Small ribosomal unit binds to the mRNA (different processes for prokaryotes and eukaryotes)
2) first tRNA (initiator) –> tRNA-met –> base pairs with the AUG start codon on mRNA
3) Large subunit binds to complete the complex
–> Binds so that the initiator tRNA is within the p-site of the ribosome
Small Ribosomal Subunit Binding in PROKARYOTES
Small ribosomal subunit rRNA binds to the:
SHINE-DALGARNO SEQUENCE on the mRNA
Shine-Dalgarno Sequence
A sequence upstream the AUG start codon on the mRNA
that small ribosomal rRNA binds to to properly position ribosome for translation
(ONLY IN PROKARYOTES)
Where is the Shine-Dalgarno Sequence located?
In the 5’ UTR region before the first start codon
Small Ribosomal Subunit Binding in EUKARYOTES
1) Small subunit interacts with the 5’ mG cap on mRNA molecule
2) Small subunit moves along mRNA “scanning” for start codon
3) Reaches start codon and initiator binds to the codon
(Not fully understood yet: just a hypothesis)
Small subunit binding position sets the…
Reading frame for translation
Once the translation initiation complex is assembled:
Elongation is ready to begin
Ribosomal “cavities”
Binding sites for tRNA: 3 of them
1) E site
2) A site
3) P site
E Site
Exit Site (LEFT)
–> Where “spent” tRNAs leave the ribosome to go get “recharged” in the cytosol
A Site
Aminoacyl Site (RIGHT )
–> Holds tRNA with next AA to be added to the peptide chain (where new charged tRNA comes in and base pairs with mRNA codon)
P SIte
Peptidyl Site (MIDDLE)
–> Holds tRNA with the growing polypeptide attached to it
–> Exit tunnel above it
Exit Tunnel
Channel in ribosome through which elongated polypeptide chain passes through the ribosome as elongation continues
Elongation Steps
1) Codon Recognition
2) Peptide Bond Formation
3) Translocation
4) Next Round
Codon Recognition Step: Elongation Phase
The anticodon of an incoming charged tRNA base pairs with the complementary mRNA codon at the ribosomal A-SITE
Peptide Bond Formation Step: Elongation Phase
rRNA catalyzes formation of a peptide bond between polypeptide from P-SITE with AA attached to the tRNA in the A-SITE
–> Peptide bond between chain and P-Site tRNA is broken –> chain then goes and attaches to the new AA in the A-Site tRNA
Translocation Step: Elongation Phase
Ribosome MOVES along the mRNA to the next codon
1) New codon at A-SITE and the site is vacant (awaiting new tRNA)
2) A-SITE tRNA with the polypeptide chain attached to it is now in the P-SITE
3) Previous P-SITE tRNA is now exiting the ribosome through the E-SITE
“Next Round” Step: Elongation Phase
Process of first 3 steps is ready to begin all over again: ribosome ready for next charged tRNA to come along
Termination begins when…
a STOP codon in the mRNA reaches the A-SITE
Termination Steps
1) STOP codon recognition
1.1) Release factor binding
2) Peptide Release
3) Ribosome recycling + Disassembly
STOP Codon Recognition + Release Factor Binding: Termination Phase
Ribosome A-SITE comes into position with a STOP codon in the mRNA
–> Release factor enters A-SITE and base pairs to the STOP codon
What does the release factor carry/cause?
Has a water molecule with it
–> Attaches the H2O to the peptide causing hydrolysis of the peptide bond from the tRNA holding it = release of protein
Peptide Release: Termination Phase
Release factor adds water to the peptide causing hydrolysis of the bond between polypeptide and tRNA molecule
–> Protein is released through the EXIT TUNNEL in the ribosome
Ribosome Recycling: Termination Phase
Everything falls apart
–> All components break apart but go on to be recycled for another round of translation
Why are release factors needed?
No naturally occurring tRNA has an anti-codon that base pairs with a STOP codon in the mRNA
Release Factor
A protein shaped like a charged tRNA that base pairs with a STOP codon in the mRNA in the A-SITE of the ribosome
–> Catalyzes the release of the polypeptide, bringing about the end of translation
Polarity of RNA vs Proteins
mRNA is made: **5’ —> 3’ **
–> Nucleotides added to free 3’ end
proteins are made: N terminus –> C Terminus
–> Free C terminus of the growing protein chain bonds to the free N-terminus of the NEW, INCOMING AA
N and C Terminals of proteins correspond to what ends of RNA
N-Terminus = 5’ end of mRNA
C-Terminus = 3’ end of mRNA
Codon is read________________
Anticodon is read _____________
Codon: Read 5’ to 3’
Anticodon: Read 3’ to 5’
Polyribosome
Many ribosomes translating the SAME mRNA sequentially and AT THE SAME TIME
What does a polyribosome allow for?
Allows for the production of many polypeptides from just one mRNA very quickly
–> Important for meeting cellular protein demands
Coordination of Transcription and Translation in Prokaryotes vs Eukaryotes
Prokaryotes = Coupled transcription and translation
(NO compartmentalization so transcription and translation can occur at the same time)
Eukaryotes = Uncoupled transcription and translation
(Temporally separated due to compartmentalization)
Monocistronic mRNA
In EUKARYOTES
–> 1 mRNA for 1 gene = production of ONE protein per mRNA
Polycistronic mRNA
In PROKARYOTES
–> 1 mRNA may contain info from multiple genes = production of multiple proteins per mRNA
What does the polycistronic nature of prokaryotic mRNA allow for?
Allows for regulation of transcription and translation of genes that are related to one another (Ex: In the same pathway)
Codons are found in ____________ NOT in ___________
Codons are in mRNA!!!
NOT IN THE DNA
