Lecture 29 - Protein Synthesis II Flashcards
What needs to be added to the 3’ end of a tRNA during processing? Why?
A CAA tri-nucleotide needs to be added because that is where the amino acids are going to be attached.
What do aminoacyl-tRNA synthetases do?
They charge tRNAs with a specific amino acid and go through a proofreading mechanism that makes sure they get it right.
What type of bond holds the tRNA to its amino acid?
High-Energy Ester Bond
Why is the genetic code said to be degenerate?
The genetic code is degenerate because it has 64 codons for only 20 amino acids.
How are certain tRNAs’ anticodon able to interact with multiple codons?
The anticodon is less faithful when interacting with the third nucleotide of the mRNA codon (3’ end of the mRNA). This third position is referred to as the wobble position. Non-complementary pairings are able to occur within the ribosome at this positon. An inosine (I) residue in this 5’ position on the tRNA can base pair with either A, U, or C in the third position of the mRNA codon. This allows several codons with the same first 2 nucleotides to interact with the same anticodon (ex. CAU and CAC for His).
Which amino acid has type associated types of tRNA? Why?
Methionine has two different tRNAs: the normal tRNAMet and the initation tRNAMet (tRNAiMet). The initation type is only used in initation of a polypeptide while the normal type is only used in elongation.
How is tRNAiMet stucturally distinct from tRNAMet?
tRNAiMet is structurally distinct in that it can bind directly to the P site of the ribosome and can interact with eIF2 in its GTP-bound active form.
What does the tRNAiMet/eIF2-GTP complex do once formed that gets it ready to interact with the mRNA?
The complex interacts with the small subunit of the ribosome (40S) associated with eIF3, eIF1, and eIF1, forming the 43S complex. eIF5 is then added to form the 43S preinitation complex, which is ready to interact with the mRNA.
Which elongation initation factor recognizes the 5’ cap? What specifically interacts?
eIF4
eIF4E interacts with the 5’ cap-binding cytoplasmic protein.
Which eIF4 subunit is an RNA helicase?
eIF4A
Which protein scans through mRNA 5’ to 3’ until it comes across a start codon (AUG)?
eIF4A
What happens to eIF2 when the start codon is recognized?
eIF2 has a change in conformation (to its inactive state) caused by its bound GTP being hydrolyzed to GDP.
What is responsible for recruiting the large subunit of the ribosome?
eIF5B-GTP
From when to when is the ribosome complex irreversibly bound to its mRNA?
It is bound from when translation is initiated to when translation is terminated.
In which site is tRNAiMet found at the start of transcription?
P Site
What happens if a tRNA finds itself in the A site and its anticodon isn’t complimentary to the mRNA codon at that site?
The tRNA diffuses out of the ribosome and another tRNA will diffuse in.
What happens when a tRNA enters the A site and its anticodon matches the codon?
EF1α, a GTP binding protein, undergoes GTP hydrolysis and is released, which changes the ribosome’s conformation such that the amino acid attached to the tRNA in the A site is in proximity to the amino acid on the tRNA in the P site so that they can form a peptide bond.
What happens after the peptide bond is formed between two amino acids in translation?
The amino acid in the P site gets transferred to the tRNA present in the A site through a peptidyl transferase reaction mediated by the 23S rRNA in bacteria and the 28S rRNA in eukaryotes. GTP-binding elongation factor EF2 (through GTP hydrolysis) allows a conformational change in the ribosome so that it moves forward one codon such that the empty tRNA is in the E site, the tRNA holding the polypeptide is in the P site, and the A site is open to accept another tRNA.
When is the tRNA in the E site forced out of the chromosome?
It is forced out when EF1α-GTP is hydrolyzed to induce a conformational change within the ribosome.
What is a ribozyme?
A ribozyme is an enzymatically active RNA molecule within the ribosome.
What happens to the discharged tRNA that was forced out of the E site?
It is recycled.
What happens when a ribosome encounters a stop codon?
The release factor eRF1 binds to the A site (as it structurally mimics aminoacyl-tRNAs) through an interaction with the stop codon. It associates to eRF3 in a GTP-bound state, which hydrolyzes GTP to GDP; thus changing the ribosome’s conformation. This results in the cleavage and the release of the polypeptide chain that was attached to the tRNA in the P site, since there is no other amino acid to form a new peptide bond with.
Why do prokaryotes have two translation termination release factors?
One of them (RF1) binds to UAG or UAA while the other (RF2) binds to UAA or UGA.
What happens to the ribosome subunits when the polypeptide is cleaved and released? Why?
The subunits of the ribosome actively dissociate because major changes in the complex occur.
What happens to the small ribosomal subunit when it dissociates after termination?
It immediately associates with eIF1, eIF1a, and eIF3 (just like in the beginning) so that the two subunits can’t come back together until the initation condition are right (AUG is found).
How does mRNA circularity help increase the rate of translation?
Since mRNA is cirucular, the end of a mRNA after translation is close to the start of the same mRNA. The ribosomal sbunits released after termination are close to the start site so that they can initate translation again.
What mediates mRNA circularity?
mRNA circularity is mediated through the interaction of eIF4G and the cytoplasmic poly A binding protein (PABPC) present at the 3’ poly A tail.
What can you identify if you subject the ribosomal complexes to sedimentation (running through a density gradient during rate-zonal centrifugation)?
You can distinguish between the different ribosomal subunits. Specifically, you can identify the non-bound subunits, the preinitation complex, the initation complex, and the competent ribosome.
