Lecture 29 - Protein Synthesis II

Question 1

What needs to be added to the 3’ end of a tRNA during processing? Why?

Answer 1

A CAA tri-nucleotide needs to be added because that is where the amino acids are going to be attached.

Question 2

What do aminoacyl-tRNA synthetases do?

Answer 2

They charge tRNAs with a specific amino acid and go through a proofreading mechanism that makes sure they get it right.

Question 3

What type of bond holds the tRNA to its amino acid?

Answer 3

High-Energy Ester Bond

Question 4

Why is the genetic code said to be degenerate?

Answer 4

The genetic code is degenerate because it has 64 codons for only 20 amino acids.

Question 5

How are certain tRNAs’ anticodon able to interact with multiple codons?

Answer 5

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).

Question 6

Which amino acid has type associated types of tRNA? Why?

Answer 6

Methionine has two different tRNAs: the normal tRNAMet and the initation tRNA^Met (tRNA_i^Met). The initation type is only used in initation of a polypeptide while the normal type is only used in elongation.

Question 7

How is tRNA_i^Met stucturally distinct from tRNA^Met?

Answer 7

tRNA_i^Met 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.

Question 8

What does the tRNA_i^Met/eIF2-GTP complex do once formed that gets it ready to interact with the mRNA?

Answer 8

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.

Question 9

Which elongation initation factor recognizes the 5’ cap? What specifically interacts?

Answer 9

eIF4

eIF4E interacts with the 5’ cap-binding cytoplasmic protein.

Question 10

Which eIF4 subunit is an RNA helicase?

Answer 10

eIF4A

Question 11

Which protein scans through mRNA 5’ to 3’ until it comes across a start codon (AUG)?

Answer 11

eIF4A

Question 12

What happens to eIF2 when the start codon is recognized?

Answer 12

eIF2 has a change in conformation (to its inactive state) caused by its bound GTP being hydrolyzed to GDP.

Question 13

What is responsible for recruiting the large subunit of the ribosome?

Answer 13

eIF5B-GTP

Question 14

From when to when is the ribosome complex irreversibly bound to its mRNA?

Answer 14

It is bound from when translation is initiated to when translation is terminated.

Question 15

In which site is tRNA_i^Met found at the start of transcription?

Answer 15

P Site

Question 16

What happens if a tRNA finds itself in the A site and its anticodon isn’t complimentary to the mRNA codon at that site?

Answer 16

The tRNA diffuses out of the ribosome and another tRNA will diffuse in.

Question 17

What happens when a tRNA enters the A site and its anticodon matches the codon?

Answer 17

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.

Question 18

What happens after the peptide bond is formed between two amino acids in translation?

Answer 18

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.

Question 19

When is the tRNA in the E site forced out of the chromosome?

Answer 19

It is forced out when EF1α-GTP is hydrolyzed to induce a conformational change within the ribosome.

Question 20

What is a ribozyme?

Answer 20

A ribozyme is an enzymatically active RNA molecule within the ribosome.

Question 21

What happens to the discharged tRNA that was forced out of the E site?

Answer 21

It is recycled.

Question 22

What happens when a ribosome encounters a stop codon?

Answer 22

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.

Question 23

Why do prokaryotes have two translation termination release factors?

Answer 23

One of them (RF1) binds to UAG or UAA while the other (RF2) binds to UAA or UGA.

Question 24

What happens to the ribosome subunits when the polypeptide is cleaved and released? Why?

Answer 24

The subunits of the ribosome actively dissociate because major changes in the complex occur.

Question 25

What happens to the small ribosomal subunit when it dissociates after termination?

Answer 25

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).

Question 26

How does mRNA circularity help increase the rate of translation?

Answer 26

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.

Question 27

What mediates mRNA circularity?

Answer 27

mRNA circularity is mediated through the interaction of eIF4G and the cytoplasmic poly A binding protein (PABPC) present at the 3’ poly A tail.

Question 28

What can you identify if you subject the ribosomal complexes to sedimentation (running through a density gradient during rate-zonal centrifugation)?

Answer 28

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.