Traditional regulation (L16)

Question 1

In initiation of translation in eukaryotes, what are the first 2 initation factor (IF) to bind?

Answer 1

eIF2 binds to GTP and it binds to the charged Met-tRNA. Then other eIFs (a cluster of them) will bind that GTP and the complex will associate w/ the ribosome subunit 40S at the P site.

Question 2

In initiation of translation in euk., how is the start codon (AUG) searched for?

Answer 2

The complex of the small ribosomal subunit (40S), Met-tRNA, eIFs-GTP will then associat w/ eIF4E (which is the cap binding protein) and other eIFs and bind mRNA.

Other initiation factors use ATP to unwind (RNA helicase) mRNA in search of AUG.

Question 3

What happens when AUG is found in initiaton of translation in eukaryotes?

Answer 3

The large ribosomal subunit associates and GTP is hydrolyzed and the eIFs leave. So all that is left in the P site is the Met-tRNA.

Question 4

What are the 3 steps of translation elongation?

Which of the 3 steps is the only that doesn’t require GTP?

Answer 4

1. EF1α-GTP facilitates AA-tRNA to bind to the A site and is released as EF1α-GDP + Pi
2. Peptidyl transferase causes AA(s) in P site to form a peptide bond w/ AA in A site (NO GTP)
3. EF2-GTP comes in and expends GTP, causing the tRNA in the P site to leave the ribosome, and the AAchain-tRNA to move to the P site and EF2 + GDP + Pi leave and there is an empty A site.

NOTE: 2 GTPs are hydrolyzed w/ the addition of each AA.

Question 5

Describe the recylcing of EF1 in eukaryotes.

Answer 5

1. EF1α-GTP helps AA-tRNA bind to A site, and is released as EF1α-GDP + Pi
2. EF1α binds to ßγ subunits, releasing GDP
3. GTP binds the α subunit of EF1α-ßγ, and ßγ subunits are released
4. EF1α-GTP is ready for another round (ie repeat)

Question 6

Describe termination of translation

Answer 6

Once a stop codon is reached, release factors enter the A site and catalyze the hydrolysis of the peptide chain from the tRNA to be released from the ribosome.

Question 7

What is a polysome?

Answer 7

A complex of one mRNA and multiple ribosomes translating simultaneously on ONE ORF. This is in eukaryotes b/c eukaryotic messages are monocistronic

Question 8

How is initiation of translation in prokaryotes different than in Eukaryotes?

Answer 8

1. Not cap-dependent. 16S fRNA recognizes shine-delgarno sequence which lies upstream to start codon.
2. The start Met AA is formylated (ie formyl is added by formylase) so it becomes fMet-tRNA
3. Porkaryotes only have 3 IFs, while eukaryotes have many more

Question 9

How does EF-G (in prokaryotes) and EF2 in eukaryotes manage to enter the ribosome?

Answer 9

They have ß-strand domains that mimic tRNA structures which trick the ribosome into allowing them to enter the A-site, hydrolyze GTP, leading to the transposition of the tRNAs

Question 10

How does the secondary structure differ b/w prokaryotic EFG and Ribosome Release Factor (RRF)?

Answer 10

EFG has ß-strand domain that mimics tRNA while RRF has α-helices that do that.

NOTE: This reveals an independent evolutionary origin

Question 11

Where does the diphtheria toxin come from?

Answer 11

It is a protein secreted by the gram (+) bacterium Corynebacterium diphtheriae but the gene of the protein was brought into the bacterial cell by an infecting bacteriophage

Question 12

How does the Diphtheria toxin work to cause cell death?

Answer 12

1. The B-subunit binds to a cell surface receptor, facilitating the entry of the A-subunit into the cell.
2. The A-subunit catalyzes a rxn in which ADP-ribose (ADPR) of NAD is covalently attached to diphthamide (a modified histidine) of EF2. This is called ADP-ribosylation of EF2.
3. This inhibits EF2, which inhibits protein synthesis, leading to cell death.

Question 13

How would one use the diphtheria toxin to its advantage?

Answer 13

Replace the B-domain with a protein that targets an unwanted protein.

Question 14

Describe translational regulation of FERRITIN synthesis

Answer 14

1. Ferritin protein binds iron and STORES it in cells when there is EXCESS IRON
2. With normal or low lvls of Fe, the Iron Response Element Binding Protein (IRE-BP) binds the Iron Response Element (IRE) (an RNA hairpin loop at 5’ end of Ferritin mRNA) to prevent translation of Ferritin mRNA.
3. High lvls of Fe, Fe binds IRE-BP dissociating it form the IRE, and translation occurs, and Ferritin is produced to store Fe in cells.

Question 15

Describe the regulation of the TRANFERRIN-receptor mRNA.

Answer 15

1. Transferrin-receptor protein transports Fe into the cell in response to LOW IRON.
2. The IRE/IRE-BP is in the 3’ end of the Transferrin-receptor mRNA, which when IRE-BP is bound (when Fe is low), prevents the mRNA from being degraded, which will cause translation of Transferrin-receptor to get more Fe into the cell
3. When Fe is high, the Fe will bind IRE-BP and dissociate it from the IRE, and the mRNA will be degraded, preventing synthesis of Transferrin-receptor

Question 16

Describe the regulation of eIF2 by Heme.

Answer 16

1. Heme prevents phosphorylation of eIF2 by Heme kinase, which phosphorylates eIF2 to inactivate it.
2. Thus active eIF2 can initiate the synthesis of the GLOBIN protein.
3. If there is low Heme lvls, Heme kinase will be active, and eIF2 is phosphorylated and rendered inactive, so globin is not produced.

SUMMARY: High heme lvls, globin is made;

Low heme lvls, globin is NOT made.

Question 17

What do microRNAs do and how are they synthesized?

Answer 17

They repress translation of SPECIFIC genes.

1. The are transcribed and form mRNA hairpin structures that are separated by DROSHA cleaving.
2. They are exported outside of nucleus and processed by DICER, where they are not a hairpin loop anymore but a single stranded piece of mRNA.
3. The RISC complex forms and the microRNA can now bind imperfectly to target mRNA (b/c they have a kink) and repress it.

Question 18

How is siRNA made?

How is it different from microRNA?

Answer 18

synthetic dsRNA is denatured(unwound) and the single siRNA enters the RISC complex and represses the target mRNA.

microRNA is natural, and it has a kink so it bind imperfectly, while siRNA is made in a lab and binds its target mRNA perfectly.

Question 19

How does the basic premise of I-cell disease (mucolipidosis II) work?

Answer 19

It is a disorder of protein targeted, where lysosomal enzyme proteins are not sorted properly from the Golgi to the lysosomes and end up being secreted from the cell. The lysosomes build up and there is a storage of clogged materials that can’t be digested and death results before age 8.

Question 20

What is the net energy used in addition of each AA?

Answer 20

2ATP, 2GTP.