Lecture 04 - Eukaryotic Translation

Question 1

What are proteins

Answer 1

the mini tools/molecular machines that carry out what we term functions in the cell

Question 2

How much of a mammalians cell energy is used for protein translation

Answer 2

30%

Question 3

What are the four major stages of protein translation

Answer 3

1. load amino acids onto tRNAs
2. Initiation
3. Elongation
4. Termination

Question 4

Why do tRNAs, elongation factors and termination factors all have similar structures

Answer 4

because they all need to fit in to the A-site

Question 5

Why is translation initiation more complex in eukaryotes than prokaryotes

Answer 5

it requires more regulation

Question 6

How many and what initiation factors are required for prokaryotic initiation

Answer 6

three
IF1, IF2, IF3

Question 7

How many and what initiation factors are required for eukaryotic initiation

Answer 7

nine
eIF1, 2, 3, 4A, 4B, 4C, 4D, 5, 6

Question 8

What are the four complexes of intitiation

Answer 8

1. 43S pre-initiation complex
2. eIF4-mRNA complex
3. 48S initiation complex
4. 80s elongation complex

Question 9

What occurs during the 43S pre-initiation complex

Answer 9

the complex is put together mRNA

Question 10

What occurs during the eIF4-mRNA complex

Answer 10

mRNA is prepped so that it is positioned properly when it binds the 43C PIC

Question 11

What occurs during the 48S initiation complex

Answer 11

the ribosome binds to the mRNA and scans for the start codon

Question 12

What occurs during the 80S elongation complex

Answer 12

60S is bring in to start elongation

Question 13

What components make up the 43S pre-initiation complex

Answer 13

40S ribosomal subunit, eIF1 and eIF1A, eIF2-GTP-Met-tRNA, eIF3, eIF5

Question 14

What is step one of the 43S pre-initiation complex

Answer 14

prepping the ribosome

Question 15

How is the ribosome prepped

Answer 15

binding of eIF1 and eIF1A causes a conformational change that opens up the channel allowing mRNA access as well as blocking the A and E sites from tRNAs

Question 16

What site does eIF1A bind to

Answer 16

the A site

Question 17

What site does eIF1 bind to

Answer 17

the E site

Question 18

What is the purpose of the eIF2 ternary complex

Answer 18

it delivers the Methionine initiator to the 40S subunit

Question 19

What is eIF2B

Answer 19

a quanine exchange factor that triggers the reloading of eIF2 with Methionine initiator

Question 20

What is unique about the methionine initiator

Answer 20

it is structurally different than methionine elongator, they cannot be interchanged, low levels of the initiator reduces translation (regulatory)

Question 21

What is eIF3

Answer 21

the largest initiation factor (13 subunits), it is used to stabilize the 43S PIC and shields the entry and exit

Question 22

What is eIF5

Answer 22

a GTPase activating protein that activated eLF2, it is usually blocked to prevent premature hydrolysis of the GTP

Question 23

What is a UTR

Answer 23

untranslated region

Question 24

What is the purpose of a 5’UTR

Answer 24

regulation of translation

Question 25

Where is a UTR

Answer 25

after the 5' cap and before the Poly-A-tail

Question 26

What are the three parts of eIF4

Answer 26

eIF4A, eIF4E, eIF4G

Question 27

What is eIF4A

Answer 27

an RNA helicase that unwinds the mRNA hairpin

Question 28

What is eIF4E

Answer 28

a cap binding protein

Question 29

What is eIF4G

Answer 29

the scaffold that binds eIF3 and the polA binding protein

Question 30

What is eIF4B

Answer 30

a factor that enhances the helicase activity of eIF4A

Question 31

What is eIF4 used for

Answer 31

the circulation of mRNA

Question 32

How does eIF4 cause the circulation of mRNA

Answer 32

eIF4G binds both eIF4E and polyA binding protein which leads to the circularization

Question 33

How is mRNA recruited onto the 43S ribosome subunit

Answer 33

eIF4G interacts with eIF3 (on the ribosome) and eIF4E (the cap binding protein attached to the mRNA)

Question 34

How does the ribosome find the initiation site

Answer 34

after binding at the 5' cap it then scans from 5' to 3' along the 5'UTR to find the AUG start codon, this must fall into the P-site

Question 35

What is the Kozak Consensus Sequence

Answer 35

a sequence prior to the start codon that tells the ribosome that the start codon is near

Question 36

What are the bases in the Kozak Consensus Sequence

Answer 36

GCCRCC(AUG)G R= either A or G

Question 37

What does it mean if translation is polycistronic

Answer 37

a weak Kozak Consensus can cause the 48S to scan past the AUG site to the next site causing a different protein to be made from the same mRNA

Question 38

What is eIF5B

Answer 38

a GTPase that mediate the joining of the 60S and 40S subunits

Question 39

What change occurs to prep for the addition of the 60S subunit

Answer 39

eIF1 is kicked out during initiation which allows eIF5 to contact eIF2 which hydrolyzes GTP to GDP (it can no longer hold the mRNA in place)

Question 40

What happens as the 60S subunit binds

Answer 40

the eIFs bound to the 40S subunit dissociate away

Question 41

What eIF stays bound and why

Answer 41

eIF1A remains bound and acts as a docking site for eIF5B

Question 42

How does elongation begin

Answer 42

60S subunit stimulates the GTP hydrolysis of eIF5B which dissociates along with eIF1A, the ribosome can now move along the mRNA

Question 43

How can viruses trick the translation system

Answer 43

fake cap making proteins, cutting caps off host mRNA, hijacking the cap making machinery, structural changes

Question 44

What is a specific example of a virus tricking the translation system

Answer 44

a type IV virus that uses its RNA to shape like a tRNA, tricks the system into thinking there is something in the P site and triggers translation

Question 45

How can a virus modify the 40S subunit

Answer 45

it can use its RNA shape to open the mRNA channel without the use of initiation factors

Question 46

What are the steps of elongation

Answer 46

1. eEF1 brings tRNA to the A site, iif the tRNA anticodon matches GTP is hydrolyzed to GDP leaving the tRNA in the A-site 2. peptide bond is formed between amino acid and growing chain 3. eEF2 fits into the A site and when hydrolyzed slides the ribosome over 3 nucleotides, the now empty tRNA in the E site diffuses away, cycle continues

Question 47

What are the steps of termination

Answer 47

1. eRF1-eRF3 binds to the stop codon 2. eRF3 is hydrolyzed and breaks off 3. energy is used to kick eRF1 further into the A site which initiates a fake amino transferase 4. sometimes the polypeptide just falls off, if not ABCE1 helps kick it off 5. ABCE1 pulls apart the subunits by further wedging in eRF1 and acts as a block so the subunits cannot reaggregate

Question 48

Why do many antibiotics target both prokaryotic and eukaryotic ribosomes

Answer 48

they are similar in structure and function, since the process of translation is so energy draining it is evolutionary conserved if there are big changes made the system will not work

Question 49

Why do tetracyclines only inhibit prokaryotic translation

Answer 49

it tends to bind to the outer edges of the ribosome which have more ability to change causing more differences between prokaryotic and eukaryotic

Question 50

How do tetracyclines work

Answer 50

binds to the small subunit and blocks tRNAs from entering the A site