Exam 3 -L17 Flashcards
In what direction does the ribosome move?
5’ -> 3’ on the mRNA strand
What two roles does the tRNA do?
- Reads the codon
2. Carries the AA
What is the Wobble Hypothesis?
It states that the first two codons always base-pairs with the tRNA strongly, while the third one is a weak pairing.
The first NT on the anticodon is in this flexible domain.
What is an advantage of the Wobble Hypothesis?
Kinetic Advantage
tRNA can dissociate quicker.
What feature is on the acceptor stem of a tRNA?
It has the sequence CCA with a free 3’OH group where the AA will be bound to.
What is the goal of an aminoacyl-tRNA synthetase reaction?
The goal is to activate an AA by forming an ester linkage with the correct tRNA.
What is the first step in charging an amino acid?
- Energize the Amino Acid by adding in ATP.
AA + ATP -> AA-AMP + PPi
What is the second step in charging an amino acid? What is unique about this step?
- Transferring the activated AA to the correct tRNA.
Aminoacyl-AMP + tRNA -> Aminoacyl-tRNA + AMP
It has two different classes of reactions.
What are the classes of aminoacyl-tRNA synthetase categorized by? (4)
- Monomer or Oligomer
- Mechanism
- General Feature of the Amino Acid
- Short stretches of amino acid similarity
What are some features of Class I aminoacyl-tRNA synthetase.
Larger, more hydrophobic amino acids.
The enzyme is a monomers.
Arg, Cys, Gln, Ile, Leu, Met, Trp, Tyr, Val.
What are some features of Class II aminoacyl-tRNA synthetase ?
Smaller, more hydrophillic amino acids.
The enzyme is a polymer.
Ala, Asn, Asp, Gly, His, Lys, Phe, Pro, Ser, Thr.
What is the mechanism for a Class I aminoacyl-tRNA synthetase ?
- 2’OH attacks AA.
2. 3’ OH attacks AA on the 2’OH. (Transesterifcation).
What is the mechanism for a Class II aminoacyl-tRNA synthetase ?
- 3’ OH attacks AA.
What is the difference in mechanisms between class I and class II aminoacyl-tRNA synthetase ?
The 3’ OH in Class II attacks start at the AA, but in Class I, the 2’OH attacks first then the 3’ OH.
Class I has an extra step.
Remember, this is all happening at the Adenine (CAA) end of the 3’ OH of a tRNA molecule.
Does the reaction involving aminoacyl-tRNA synthetase require energy?
Yes!
How many domains does the enzyme aminoacyl-tRNA synthetase have?
- Catalytic Domain
2. Variable Domain
What does the catalytic domain do?
- Interacts with tRNA’s 3’ End
2. Recognizes and binds to the correct AA
What does the variable domain do?
Interacts with the specific bases on tRNA that identify that specific tRNA.
What are the three types of editing function of the aminoacyl-tRNA synthetase?
- Size exclusion
- Editing Pre-Transfer
- Editing Post-Transfer
Where does transcription occur in eukaryotes?
In the nucleus.
How does mRNA get exported out of the nucleus?
Protein factors bind to the mRNA, taking it to a nuclear pore.
Once there, a protein outside of the bore, Dbp5, acts like a helicase and binds to the mRNA, pulling it out into the cytoplasm.
What is the recognition site for a ribosome when dealing with prokaryotes?
Shine-Dalgarno Sequence
What is the recogniztion site for a ribosome when dealing with eukaryotes?
5’ End Methylated Guanine Cap.
What subunits make up a ribosome for a prokaryote?
30S + 50S = 70S
What subunits make up a ribosome for a eukaryote?
40S + 60S = 80S
What region within the 30S subunit in prokaryotes binds to the Shine-Dalgarno sequence?
16S region interacts with the 30S subunit.
What region within the 50S subunit in prokaryotes does the peptidyl-transfer?
23S region within the P site interacts with tRNA to form a peptide bond between adjacent amino acids.
What do GAPs do?
They hydrolyze GTP.
GTP -> GDP + Pi
What do GEFs do?
GEFs allow for GDP to dissociate, with the addition of a new GTP. Recycling.
What functions as a GAP in prokaryotic translation?
The ribosome.
What is the very first thing that gets read during the initiation of translation in prokaryotes?
Correct registration of mRNA with a ribosome requires the alignment of a PYRIMIDINE RICH sequence on the 3’ END OF THE 16S RNA (30S subunit) with a PURINE RICH sequence of the 5’ END of mRNA (Shine Dalgarno)
What five things make up the 30S initiation complex?
- 3 Initiation Factors (IF1, IF2, IF3)
- GTP
- fMet-tRNA^Met
- mRNA
- 30s Subunit
In prokaryotes, what has to occur to the tRNA for initiation to occur?
The first tRNA to initiate translation has to have the addition of a formyl group. fMet-tRNA
It also has a normal Met-tRNA as well.
Do Eukaryotes use fMet-tRNA?
No, we use a normal Met group, but a special tRNA.
When does IF3 bind and what does it do?
- It is the first to bind.
IF3 binds to the E site.
Prevents tRNA from binding to that site and also prevents the 50S subunit from coming in too early.
When does IF1 bind and what does it do?
- After IF3 has bound.
IF1 binds to the A site.
Prevents tRNA from binding to that site.
When does mRNA bind?
- It binds after IF3 and IF1 have bound.
When does IF-2-GTP bind and what does it do?
- After mRNA has bound to IF1 and IF3
It binds to IF1 and slightly in the P site. It recruits the tRNA to the P site.
When does fMet-tRNA bind and what does it do?
- After IF2-GTP has bound.
It binds to the P site and it was recruited by IF-2-GTP.
It will be the starting tRNA .
When does the 50S subunit come in and what does it do?
- The 50S subunit comes in after the Pre-Initiation Complex has assembled right after
When it comes in, it will displace IF3.
What happens after the 50S has displaced IF3?
- 50S will interact with IF-2-GTP, hydrolyzing it and causing a conformational change that will dissociate IF1 and IF2.
This will complete the assembly of the 70S initiation complex.
What parts of eukaryotic mRNA is involved in initiation of translation?
The 5’ cap and the 3’ Poly-A-Tail
The 5’ Cap is bound by what translation factor?
EIF4E, part of the EIF4F complex.
The tail of the mRNA is going to be bound by what?
PABP will interacting with the EIF4F complex.
When does EIF1 bind and what does it do?
- EIF1 is the first to bind.
It occupies the E site and prevents the 60S complex from coming in.
When does EIF1A bind and what does it do?
- It binds after EIF1 binds.
It occupies the A site.
When does EIF3 bind and what does it do?
- It binds after EIF1A binds.
Helps with translation.
When does EIF2-GTP-tRNA bind and what does it do?
- It binds after EIF3 binds.
It brings Met-tRNA^Met to the P site with the help of EIF1 and EIF1A.
When does EIF-5B-GTP come in and what does it do? it do?
- It binds after EIF2-GTP-tRNA
It binds near the P Site.
When does EIF4F-mRNA come in and what does it do?
- It comes in after EIF-5B-GTP comes in.
It contains the mRNA.
What happens after EIF4F-mRNA comes in?
It joins in the the other subunit to scan for the start codon.
What happens after the pre-initiaton complex finds the start codon?
It will hydrolyze EIF2-GTP and EIF-5B-GTP allowing for the 60S subunit to bind, forming the 80S ribosome subunit.
What does the PABP interact with to form a loop?
EIF4
Explain elongation in translation. (3)
- EF-Tu-GTP binds to an aminoacyl-tRNA.
- Complex binds to the A-site, GTP is hydrolyzed and the EF-Tu-GDP complex dissociates.
- EF-Tu-GTP gets recycled via Ef-Ts.
Explain how Ef-Tu-GDP is recycled.
- EF-Ts comes in and displaces the GDP on Ef-Tu-GDP.
2. Then a GTP comes in and displaces the EF-Ts Forming once again, EF-Tu-GTP
How does hydrolyzing EF-Tu-GTP allow for elongation?
It causes a conformation change that stimulates GTPase activity.
Where does the peptide bond formation occur>
It occurs at the 23S region of the 50S.
How is the peptide bond formed during translation?
The amino group of the amino acid in the A site acts as a nucleophile.
It attacks the carbonyl group of the amino acid in the P site.
How does translocation occur? (movement of the tRNA into a different site)
A different factor, EF-G-ATP, similar in structure to a tRNA, enters the pocket at which the EF-Tu just left and binds to it.
This binding to the amino acid in the A site will cause GTPase activity where it will become EF-G-ADP.
A conformational change will be induced, pushing the tRNA from the A site into the P site.
How is translation terminated?
Proteins known as Release Factors recognize the stop codon in the A region of the ribosome.
How do release factors initiate transcription?
The RF transform the peptidyl transferase site into a hydrolase, cleaving the peptidyl chain from the tRNA carrier.
What are the release factors in prokaryotes? Which codons do they read.
RF1 -> UAA and UAG
RF2 -> UAA and UGA
RF3 -> Releases the RF
What are the release factors in euk?
ERF -> recognizes all stop codons.
