Week 4: Translation Flashcards
Why is the RNA code considered “degenerate”?
The triplet codes for 64 possible combinations because it is not 1 codon per amino acid (not efficient). This is not random, because most of the variation that promotes this redundancy comes from the third codon position (i.e. UCU, UCC, UCA and UCG all code for serine)
How do viruses use frameshifting to their benefit?
They use overlapping reading frames for the same sequence, meaning the same sequence can code for different AAs based on where the reading frame begins.
What is the start codon and what are the three stop codons?
AUG is the start codon and UAA, UGA and UAG are stop codons (third base isn’t so important, usually)
What does the fidelity of ultimate protein expression depend on? What is the largest source of error?
RNA transcription
reliability of aminoacyl-tRNA synthetase activity (enzyme that binds a tRNA to its aminoacyl group)
proper codon-anticodon pairing (largest source of error)
reliability of ribosomes to shift mRNA within the apparatus to avoid frameshifts
What enzyme attaches the amino acids to the tRNA? What end of the tRNA attaches the amino acid? Where is the “wobble” position on the tRNA?
The aminoacyl-tRNA synthetase attaches tRNAs to their respective amino acids at the 3’ end of the molecule. The 5’ end of the anticodon arm section acts as the “wobble” position (this will be the last one “read” by the tRNA, and represents the third RNA in a given codon)
What amino acid confers the most “flexibility” to the wobble position?
Hypoxanthine, or “I” can bind to A, U or C on the codon. U (bind A and G) and G (binds C and U) have the next most flexibility, whereas A and C have the least.
Why are there only 32 tRNAs?
There are 32 “sets” of codons that share the first two letters, but vary in the third letter, with U/C grouped and A/G grouped
What are the steps of aminoacyl tRNA activation?
(1) The amino acid and ATP bind to an aminoacyl-tRNA synthetase (there can be more than one of these for each kind of AA)
(2) A pyrophosphate is cleaved as the aminoacyl group binds to the a-phosphate of ATP, forming pyrophosphate (cleaves immediately, producing energy, and meaning the formation of each activated tRNA requires 2 ATP), and forming the aminoacyl-AMP molecule
(3) 3’ OH of the corresponding tRNA moves in, binding the aminoacyl group and forming an activated aminoacyl-tRNA
What are the large and small subunit sizes in eukaryotes and bacteria?
Eukaryotes:
Total = 80S, Large = 60S, Small = 40S
28S rRNA of large subunit responsible for peptidyltransferase activity
18S rRNA of small subunit help bring the mRNA into place and align the start codon
Prokaryotes:
Total = 70S, Large = 50S, Small = 30S
23S rRNA of large subunit responsible for peptidyltransferase activity
16S rRNA of small subunit help bring the mRNA into place and align the start codon
What are the similarities in prokaryotic and eukaryotic ribosomes?
Both have A (aminoacyl tRNA) and P (peptidyl-tRNA) sites formed by a combination of large and small subunits.
Only prokaryotes have an E (empty tRNA) site within the large (50S) subunit that is not present in eukaryotes
Prokaryotic translation is started by an fMet-tRNAfMet whereas eukaryotes begin translation with Met-tRNAMet.
The initiation of translation in prokaryotes and eukaryotes always begins with the initiator tRNA beginning at the P site.
What is the Shine-Dalgarno sequence?
It is the prokaryotic mRNA sequence that helps secure it to the 30S subunit of the ribosome. It binds to the 16S subunit, more specifically.
What are the steps of initiating mRNA binding in prokaryotes?
(1) The 30S subunit binds IF-1 and IF-3, then the Shine-Dalgarno mRNA sequence binds to the 16S rRNA
(2) IF-2-GTP binds the 30S subunit and recruits fMet-tRNA to the P site, which base pairs with the start codon
(3) GTP is hydrolyzed and IF1, 2 and 3 dissociate as the 50S subunit moves in
What are the initiation steps in eukaryotes?
(1) eIF1, 3, and 1A bind to the 40S subunit and form a preinitiation complex.
(2) eIF2-GTP moves in to fill the E site and Met-tRNAiMet moves in to fill the P site. mRNA is NOT bound at this point.
(3) A cap-binding complex binds to the 5’ cap of mRNA and helps it move into place on the subunit.
(4) eIF4F uses ATP hydrolysis to slide the mRNA within the ribosome untnil the start codon properly aligns with the tRNA anticodon in the P site. This differs from prokaryotic mRNA, which is immediately positioned with the start codon in place, whereas the eukaryotic version requires ATP hydrolysis to slide the start codon into place.
(5) The whole complex is now called the 48S subunit. All the initiation factors dissociate to allow the large subunit to bind, creating the eukaryotic initiation complex.
What is the rate-limiting step in the formation of the initiation complex in eukaryotes, and why is this?
Hydrolysis of the GTP bound to GTP-eIF2 is rate-limiting. This is because Met-tRNA is in abundance. Because GTP-eIF2 brings the Met-tRNA into place, and requires hydrolysis, this step is rate-limiting.
Enzyme activity affecting the eIF2 state (bound to GTP vs GDP + Pi) can be regulated. Specific enzymes are used to regenerate GTP on eIF2.
Describe how elongation begins
An aminoacyl tRNA bound to EFTu-GTP (eEF1a in eukaryotes) binds to the A site of the ribosome.
GTP hydrolyzes, releasing EFTu-GDP
What is peptidyltransferase and how does it direct tRNA movement along the ribosomal sites?
Peptidyltransferase is the small subunit of the ribosome (28S in eukaryotes, 23S in prokaryotes), and it helps catalyze the nucleophilic attack of the incoming AA (in the A site) on the carbonyl of the existing chain (in the P site).
Once the peptide bond forms, the P site tRNA is empty. In prokayotes, it shifts to the E site and is ejected. In eukaryotes, it is directly ejected from the ribosome.
How does termination occur?
When a stop codon is presented in the A site, it is recognized by a release factor RF (pro) or eRF/eRF1 (eu). This release factor hydrolyzes the peptide from the peptide-tRNA in the P site, releasing it from the ribosome.
Then, RF3 hydrolyzes the GTP, and this energy release of the EFG-GTP cleavage provides the energy to allow the ribosome to release the de-acetylated tRNA from the P site, and the RF from the A site.
Finally, ribosome recycling factor (RRF) acts with EFG and IF3 (eIF1 in eu) to break the ribosome into it’s respective subunits.
