6. Translation

Question 1

why is the 5’ cap important in eukaryotes?

Answer 1

allows 5’ and 3’ end to be close together –> for transcription initiation

Question 2

how is the ribosome recruited to mRNA?

Answer 2

5’ cap is recognized by Eukaryotic Initiation Factor 4E, i.e. cap-binding protein

Question 3

what does IRES stand for?

Answer 3

Internal Ribosome Entry Site

Question 4

if the poliovirus genome has the VpG protein but no cap structure, how can translation be initiated?

Answer 4

by CAP-INDEPENDENT TRANSLATION

Question 5

Describe cap-independent translation

Answer 5

the poliovirus 5’ UTR contains an Internal Ribosome Entry Site that allows internal initiation

Question 6

how do ribosomes recognize IRES?

Answer 6

based on RNA secondary structure

Question 7

can you predict IRES from primary RNA sequence?

Answer 7

technically, the ribosome recognizes specific secondary structures BUT if you see a long 5’ UTR that could form stem loops and other specific structures, can deduce that it involves IRES

Question 8

what are the eIF requirements for 5’-end-dependent initiation?

Answer 8

all eIFs involved

Question 9

what are the eIF requirements for type 1 or 2 IRES?

Answer 9

all eIFs involved except eIF4E

Question 10

describe type 1/2 IRES

Answer 10

virus causes cleavage at eIF4G lets it shut down cellular replication so only viral replication can occur

Question 11

what are the eIF requirements for hepatitis C virus IRES?

Answer 11

only requires eIF2 and eIF3

Question 12

what are 6 mechanisms used by viruses to manipulate the translation?

Answer 12

1. ribosome shunting
2. methionine-independent initiation
3. leaky scanning
4. re-initiation
5. suppression of termination
6. ribosomal frameshifting

Question 13

describe 1. RIBOSOME SHUNTING
- how does it work
- why is it helpful for the virus

Answer 13

ribosomes bypass/shunt over parts of the 5’ untranslated region to reach the start codon or bypass a stable secondary structure in the mRNA

helpful for the virus bc normally it would get stuck and have to unwind the secondary structure but this reduces the need for unwinding

Question 14

describe 2. METHIONINE-INDEPENDENT INITIATION for the Cricket Paralysis Virus

Answer 14

the CRPV IRES mimics the Met-initiator tRNA (Met-tRNAi –> allowing the virus to recruit 80S ribosomes without any eIFs or Met-tRNAi

Question 15

describe 2. METHIONINE-INDEPENDENT INITIATION for the Turnip Yellow Mosaic Virus

Answer 15

TYMV mimics tRNA and binds to valine on the P site of the ribosome

Question 16

describe 3. LEAKY SCANNING
- which type of viruses
- how does it work
- why is it helpful for the virus

Answer 16

• used by PARAMYXOVIRUSES
• some ribosomes initiate translation at non-optimal codon via diff reading frams, diff start codons (ex. ACG 81, AUG 104 - bad) –> this tricks the ribosome to read the mRNA differently
• this is helpful for letting the virus pack lots of info in its mRNA and encode several proteins on the same mRNA

Question 17

which method of translation manipulation is used with leaky scanning? why?

Answer 17

ribosome shunting –> allows for the other 2 open reading frames to be reached

Question 18

describe 4. RE-INITIATION

Answer 18

• once ribosome reaches stop codon, the 40S subunit stays bound to the mRNA and the 60S subunit detaches then rejoins to allow RE-INITIATION for expression of a downstream ORF

Question 19

describe 5. SUPPRESSION OF TERMINATION (READTHROUGH) in HIV

Answer 19

• in HIV, a UAG codon (stop codon) is suppressed 10% of the time to allow for synthesis of Gag-Pol fusion protein because the mRNA forms a pseudoknot –> ribosome pauses, allowing more time for a suppressor tRNA to be charged and forming Gag-Pol fusion protein

Question 20

describe 6. RIBOSOMAL FRAMESHIFTING
- how does it work
- what is required for it to work
- why is it needed (2)

Answer 20

• ribosomes pauses, slips back and shifts into a different reading frame (+/- 1)
• requires a sequence that promotes ribosome slippage and the tRNA to move by 1 base
• needed bc it helps synthesize protein encoded in a different reading frame AND can bypass a stop codon

Question 21

what is an example of ribosomal frameshifting being required?

Answer 21

In retroviruses, Pol may overlap with Gag so it requires diff reading frame via ribosomal framshifting

Question 22

what is the main purpose of translation tricks?

Answer 22

to let viruses pack A LOT of info into the mRNA –> ex. 1 mRNA can make multiple proteins, fusion proteins, etc.

Question 23

how do viruses affect host protein synthesis?

Answer 23

turn OFF host protein synthesis

Question 24

why do viruses turn off host protein synthesis?

Answer 24

to prevent infected cells from mounting an antiviral response

Question 25

explain this graph:

Answer 25

• uninfected cells increase their rate of protein synthesis at constant rate
• in infected cells: host protein synthesis decreases quickly
• then reaches a point where host protein synthesis is turned off
• then viral protein synthesis increases

Question 26

explain this SDS-PAGE

Answer 26

cellular proteins are made at the beginning, then at 5h post infection, only viral proteins are made

Question 27

what virus is the 2A protease from?

Answer 27

poliovirus

Question 28

what are the 3 steps of the activity of poliovirus 2A protease?

Answer 28

1. poliovirus 2A protease cleaves eIF4G
2. this prevents host cap-dependent protein synthesis by bridging the cap-binding protein eIF4E to the translation initiation complex
3. host protein synthesis is inhibited but viral protein synthesis can continue

Question 29

why can virus protein synthesis continue after poliovirus 2A protease activity?

Answer 29

2A protease blocks cap-dependent protein synthesis but since poliovirus protein synthesis depends on IRES not a cap structure, the viral protein synthesis can continue

Question 30

how do viruses attack machinery for host translation?

Answer 30

inhibit host translation factors by cleaving, (de)phosphorylating, etc.