Topic 11 Translation Flashcards
What is translation
Conversion of nucleic acid code to amino acid code
What are the 3 molecules needed for translation
Messenger RNA (mRNA): information carrying molecule
that encodes the info specifying the protien sequence
Transfer rna (tRNA): amino acid adapter molecule
carried amino acids and recognizes the mRNA sequence through complimentarity
single stranded rna
Ribosome: amino acid linking complex,
catalyzes peptide bond formation and binding of ribosome to mRNA is where translation happens
What else is very important for translation
Aminoacyl TRNA synthetases: enzymes that catalyze the charging of each of the 20 amino acids to the appropriate tRNA
How does the transcript info turn into protien info
The mRNA info is read as a triplet code
Each triplet code has a 3 nucleotide codon called the GENETIC CODE
What is a codon
A sequence of 3 nucleotide that codes for a specific amino acid
What is the genetic code
How many diff combo of codons
What are the stop and start codons
What is wobble
The ordered series of codons in the mature mRNA that specify the order of amino acids
The code has 4^3 = (64) diff combinations of codons
Aug start, UGA UAA UAG stop
The wobble:
The third nucleotide in each codon can be diff but still encode the same amino acid
This is why the code is denergate
What reads the mRNA code
What are the important parts
tRNA : 5-3’ single stranded RNA folded into a cloverleaf structure by intramolecular base pairing
Anticodon loop (bottom): recognizes and base pairs with the mRNA
3’ amino acid attachment site: carries the appropriate amino acid residues based on the codon in the anticodon region
How does the tRNA mature to have that longer 3’ end
Through its ribozyme activity allowing it to cleave itself into mature tRNA
What are ribosomes
What are the structure of ribosomes in prokaryotes and eukaryotes
Ribosomes have one large and one small subunit
large has the peptidyl transferase center that catalyzes peptide bond formation
small has the decoding centre that decodes the mRNA codon (ex. The first AUG in the mRNA)
Each subunit has RNA and Protien, meaning it’s a ribonucleoprotien
Prokaryotes: small 30s large 50s
Eukaryote: small 40s large 60s
Describe the sites in the ribosome
EPA
A (aminoacyl) site: binds the incoming aminoacyl tRNA (tRNA with amino acid charged at 3’ end)
P (peptidyl) site: has the growing polypeptide chain of amino acids, the first amino acid is MET which is the n term or the protien
E (exit) site: after ribosome moves one codon down the previous deacylated tRNA is released and the new tRNA takes the polypeptide chain
Peptidyltrasnferase centre: in the large subunit catalyzes the peptide bond formation though peptidyl transferase activity
When does translation stop and why
Describe the sequential recruitment of the ribosome
Stops when the ribosome reaches the stop codon since there is no tRNA for the stop codon
The small subunit with the first met tRNA are bound first and scan across the mRNA for the start codon. Then the large subunit is recruited
What is the ORF
What polycistronic mRNA
What is monocistronic mRNA
ORF: stretch of dna or rna between the start and stop codon which is translated into protien
Polycistronc: in prokaryotes mRNA, one mRNA transcript with multiple ORF, so translation of multiple proteins happens at the same time , more efficient
Monocistronic: in eukaryotes mRNA, one mRNA transcript with one ORF so translation of one protein, slower less efficient
What are the 3 possible reading frames for the rna strand
- Start at aug
- Shift by one nucleotide
- Shift by 2 nucleotide
All make diff protien product so appropriate reading frame is important
How can you verify that your mRNA was translated with the core t open reading frame
Usually has to start at the start codon
Can do western blot to see if size has change from what was expected
What are the feature of the eukaryotic mRNA that allow translation
5’ cap: protects from degradation and recruits the ribosome to the 5’ end of the mRNA
Then the ribosome scans 5-3 until reaching start codon
Kozack sequence: purine (A or G) 3 bases upstream of the AUG start codon and a G right after increases the translational efficiency (translation still happens without it but less efficiently)
(G/A)NNAUGG
Poly A tail: 3’ end tail recruits key translation initiation factors and enhances the translational efficiency
What is the common features of tRNA
there are many types of tRNA with each attached to a specific amino acids and recognizes a specific codon(s)
75-95 ribonucleotides long
3’ end ends with CCA which is required for amino acid attachment
Some tRNA have modified nucleosides in their primary structure
Explain the modified residues in the tRNA
Regular uridine: has N1 glycosidic bond
Pseudouridine (Triton U): no N1 bond, C5 glycosidic bond
Dihydrouridine: no double bond between C5 and C6, so 2 extra H
Each are in the tRNA loops
Describe the secondary structure of tRNA
Acceptor stem: CCA 3’ site for amino acid attachment
Triton U loop (right): has pseudouridines
D loop(left): has dihydrouridines
Variable loop: variable in size in diff tRNA structures
Anticodon loop: has the 3 nt sequence that recognizes the codon on mRNA by base pairing with it
Describe the tertiary structure of tRNA and how they know which amino acids need to be added
The 3’ acceptor ends has the amino acid attachment catalyzed by amino acyl tRNA synthetase
Specific Amino acyl tRNA synthetases recognize the anticodon loop and the acceptor stem of the tRNA to add the correct amino acid to the specific tRNA
Describe the steps in attachment of an amino acid to tRNA
Adenylation of the amino acid:
the coo- of the amino acid attacks the alpha phosphate of the ATP, pyrophosphate leaves
Now amino acid is adenylated and bound to tRNA synthetase
tRNA charging:
The 3’ OH of the tRNA acceptor stem attacks the adenylated amino acid
Now amino acid attached to tRNA (tRNA charged) and AMP leaves
This make aminoacyl tRNA
How many tRNA synthetases are in organism and what do they do
Since 20 diff amino acids there are 20 sybrneatse for each amino acid for each tRNA
One specific synthetase for that amino acid attaches it to a specific trna
What are the two types of tRNA
Aminoacyl tRNA: charged tRNA bound to the a site
Peptidyl tRNA: has the growing polypeptide chain and is in the P site
What does the s mean in ribosome subunits (ex. 40s)
What determines sedimentation velocity
S = Svedberg unit
Larger s = faster sedimentation velocity (if centrifuged they settle to the bottom faster) and larger mole/size which is why they settle faster
Shape and size determine sedimentation velocity
S does not mean MW this is why 60s and 40s don’t make 100s but they make 80s
What is in the core functional domain of the ribosome
rRNA
For example in the peptidyl transferase centre and the decoding centre there is mainly rRNA
This rna is conserved across all organsims and important for the large and small subunits functions
Describe the ribosome cycle
The large and small subunits of the ribosome undergo association and dissociation during each translation
Each mRNA can be can be translated at the same time by multiple ribosomes
New amino acids are added to the c term of the growing chain
- Small subunit of the ribosome first bind to met TRNA and scan across ribosome to find Aug
- Once Aug found, large subunit comes in, met tRNA in P site
- Amino acyl tRNA comes into A site
- The met residue is transferred to the Aminoacyl tRNA , ribosome shifts, now uncharged tRNA in E site and peptidyl tRNA in P site
- Ribosome reaches stop codon and release factors come in to dissociate the ribosome complex and polypeptide
Describe the general steps of eukaryotic initiation
- met tRNA (initiator tRNA) bind to small 40s subunit
- Auxiliary factors help recognition of mRNA, expose the AUG, further recruit the large subunit
- The 40s with met tRNA scans for Aug
- 60s is recruited after the met tRNA pairs with start Aug
What are the auxiliary factors in eukaryotic initiation and their roles
Eukaryotic initiation factors (eIF)
eIF1, eIF1A, eIF3, eIF5:
block the E and A site to so met tRNA bind only to P site, and prevent 60s binding
eIF2-GTP:
Brings the met tRNA to the o site in the 40s to make the 43s preinitiation complex
eIF4:
Is a helicase that straightens out mRNA which is in secondary structure which hides the AUG
So it prepares the mRNA for recognition by the met tRNA by straighten out mRNA and exposing AUG
Once this is done the tRNA can scan for the Aug
What is special about eukaryotic initiation of translation
Atp dependent
What happen in eukaryotic initiation when the met tRNA find the Aug
The preintiation compelx moves in 5-3 direction atp dependent manner and scan for Aug
Once met tRNA base pairs, 43s complex releases eIF1, eIF2-gdp (since used energy to charge not grp anymore), eIF3, eIF4, eIF5
eIF5B-GTP:
Binds to stimulate association of 60s subunit. Binding of 60s further releases the intitation factors
Then met tRNA stay in P site until Aminoacyl tRNA comes in
Describe eukaryotic elongation
- Loading of Aminoacyl tRNA to A site
- Peptidyl transferase reaction: transfer of the growing polypeptide chain from peptidyl tRNA to Aminoacyl tRNA. Froms a peptide bond between Aminoacyl and peptidyl tRNA amino acids
- Translocation of Aminoacyl tRNA from A site to the P site by ribosome moving one codon
What is the problem with elongation and how is it fixed
Before the Aminoacyl tRNA comes into the P site, it js floating in the cytoplasm
Here it can form a peptide bond with other Aminoacyl tRNA
Prevented by elongation factor EF-TU
How does EF-TU help in elongation
EF-TU-GTP:
bind to the 3’ end of the incoming Aminoacyl tRNA to block it from making premature peptide bonds with other tRNA
Correctly positions the Aminoacyl tRNA in the A site by recognizing the correct anticodons : codon binding in the A site and allowing the tRNA to rotate to fit properly in the A site
EF-TU-GDP:
When gtp form is bound in A site it hydrolyzes to gdp form in the factor binding centre in the 60s subunit
Then EF-TU-GDP is released from tRNA
What are the three mechanisms for correct recognition of the codon with it’s appropriate tRNA anticodon and correct positioning of the amino acyl tRNA
In prokaryotes
In the small subunit there is prokaryotic 16s rRNA.
There are 2 additional h bond formed between the two adenine residues in this rRNA and the minor groove of the correct anticodon-codon base paring in the tRNA
As soon as the first two nucleotides of the tRNA anticodon bind to the first two nt of mRNA codon, this h bonding can happen to Stabilize the anticodon to the mRNA
If incorrect base pairing, this h bonding doesn’t happen and the tRNA is not stabilized at the codon
Ensures correct recognition of tRNA anti cond with mRNA codon
- EF TU GTP is only able to interact with the factor binding centre to be hydrolyzed and released when there is correct base pairing
If incorrect base pairing, stays GTP and attached to tRNA, tRNA then leaves
- only when there is correct base pairing can the tRNA rotates into the correct position to make the peptide bond
If not correct tRNA doesn’t rotate and just leaves
When does translation termination happen
When stop codons enter the A site, release factors recognize them and activate hydrolysis of the growing polypletide from the peptidyl tRNA
What are the two classes of release factors
What’s special about the RF
Class I: RF1
recognize the stop codons and triggers hydrolysis of the peptide chain from the tRNA in the P site
Only one class 1 RF in eukaryotes that recognizes all three stop codons
Class II: RF3
Stimulates the dissociation of class 1 from the ribosome after the polypeptide chain is released
Describe the process of termination
- RF1 recognizes the stop codon, binds in A site, and stimulates release of the polypeptide in the P site through its GGQ motif
Now no polypeptide in P site
- RF3-GTP binds after the poly peptide is released and displaces the class I RF
- RF3-GTP interacts with the factor binding centre of the large subunit to hydrolyze. RF3-GDP dissociates from the ribosome
- The deacylated tRNA are still in the P and E site so RRF (ribosome recycling factor) binds to the A site to recruit EF-G-GTP
- EF-G-GTP releases the uncharged tRNAs and displaces RRF from the A site
- EFG-GTP hydrolyzed to GDP form and dissociates, then ribose and mRNA are dissociated and released
Termination occurs in a
Stepwise manner
