Protein Synthesis-- Translation from RNA to Protein

Question 1

Why study protein synthesis?

Answer 1

• last opportunity for regulating gene expression
• rapid response to stimuli (transcription sometimes takes too long b/c in the nucleus)
• many pharmaceutics impact translation (i.e. antibiotics)

Question 2

STOP codons

Answer 2

UAA (U Are Annoying)
UGA (U Go Away)
UAG (U Are Gone)

Question 3

START codon

Answer 3

AUG (Methionine)

Question 4

Nonsense Mutiation

Answer 4

STOP speaking that nonsense

mutation creating stop codon

Question 5

Key Players in Translation

Answer 5

• mRNA: the code
• tRNA: the adapter
• the ribosome: the enzyme
• *** Above ONLY NEEDED for protein synthesis **
• factors: the multitude of proteins that make the system work (increase efficiency, but are not necessary for occurrence)

Question 6

5’ cap

Answer 6

• specialized nucleotide that is required for BINDING OF INITIATION FACTORS
• stability

Question 7

5’ UTR

Answer 7

sequence between 5’ cap and the initiation codon

Question 8

Coding Region

Answer 8

sequence that gets translated into protein

Question 9

3’ UTR

Answer 9

• sequence between the stop codon and the poly(A) tail

- site of key regulatory sequences

Question 10

poly(A) tail

Answer 10

untemplated polyadenylate sequence averaging ~200 nucleotides that protects mRNA from degradation and increases translational efficiency

Question 11

5’ UTR

3’ UTR

Answer 11

5’ UTR: regulation; non-coding

3’ UTR: regulation; non-coding; stability

Question 12

Aminoacylation

Answer 12

AA added to 3’

• enzymes (aminoacyl tRNA synthetases) recognize tRNAs and add appropriate AA
• requires ATP
• ** ACTIVATION; HIGH ENERGY BOND FORMED via AMP; AA attached to 3’ end ***

Question 13

Translational Fidelity

Synthesis site vs Editing site

Answer 13

identifies anticodon loop (only proper loop will fit in enzyme; SPECIFICITY OF SHAPE AND CHARGE)

• synthesis site: v high affinity for proper AA; if incorrect cannot access site unless similar in size and structure
• editing site: v high affinity for INCORRECT AA; if enters editing site it will be cleaved off

Question 14

Anticodon Loop

Answer 14

• recognizes codon
• aminoacylation adds proper AA to 3’ end of tRNA
• ATP driven

Question 15

Ribosome = Ribozyme

Answer 15

rRNA is sufficient to carry out protein formation; it is an enzyme but poor efficiency w/o additional enzymes

Question 16

Ribosome Binding Sites

Answer 16

A (aminoacyl): initial tRNA binding site for next codon
P (peptidyl): peptide bond formation
E (exit): tRNA release

Attach’ Put on bond; Exit

Question 17

Translation Cycle

Answer 17

Initiation (Initiation Factor)
Elongation (Elongation Factor)
Termination (RELIEF Factor)

Initiation and Elongation occur at the same time with different ribosomes

Question 18

Polyzome

Answer 18

mRNA + multiple ribosomes

mRNA ribosome complex

Question 19

Initiation

Answer 19

• assembly of ribosome and mRNA

- positioning of the ribosome on the start codon (AUG)

Question 20

Where does initiator tRNA enter at?

Answer 20

tRNA-MET enters at P site; all others enter at A site

** INITIATION tRNA DIFF THAN NORMAL MET tRNA (different one used during elongation) **

Question 21

eIF4E

Answer 21

Initiation

binds to the 5’ cap; recruits eIF4G

Question 22

eIF2

Answer 22

Initiation

binds and delivers initiator Met-tRNA (requires GTP)

Question 23

eIF4G

Answer 23

Initiation
scaffold protein that binds eIF4E; required for assembly of pre-initiation complex
- bound at 5’ end and interacts with 3’ end creating circular mRNA allowing subunit to attach

Question 24

Large subunit attachment

Answer 24

small subunit attaches post circularization of mRNA; once attached will scan for AUG codon; once reaches AUG large unit will come and attach

Question 25

eIF2 release

Answer 25

(eIF2 binds and delivers intiation Met-tRNA to P site) GTP hydrolysis cause eIF2 release - once released large subunit attaches

Question 26

Elongation

Answer 26

movement of the ribosome down the mRNA coordinated with aminoacyl tRNA delivery

Question 27

rRNA site Occupation

Answer 27

at any given time only TWO SITES OCCUPIED

Question 28

Initiation Steps Overview

Answer 28

1) formation of pre-initiation complex | 2) Scanning to AUG codon

Question 29

Elongation Steps Overview

Answer 29

1) delivery of aa-tRNA to A site and E site release 2) GTP hydrolysis and eEF1A release 3) eEF2 binding to catalyze translocation 4) GTP hydrolysis and eEF2 release; completion of cycle

Question 30

eEF1A

Answer 30

Elongation | binds all cononical tRNAs (all except initiator and selenocysteine tRNA)

Question 31

GTP hydrolysis is required

Answer 31

for release and promoting next step

Question 32

eEF2

Answer 32

Elongation - drags along large subunit; small unit follows; movement allows for elongation - G-protein required for ribosome translocation - if eEF2 is affected TRANSLOCATION CANNOT OCCUR, thus A site will never be empty and translation cannot proceed

Question 33

"Proofreading" during Elongation

Answer 33

occurs in the ribosomal A-site where codon/anticodon pairs are "checked" by ribosome conformation - incorrectly base-paired tRNAs preferentially dissociate

Question 34

Termination

Answer 34

eRF2 binding and peptide hydrolysis

Question 35

eRF1

Answer 35

- attaches and terminates protein synthesis - recruited when STOP codon is recognized * ** NO tRNA for STOP codons *** (tRAN mimetic and catalyzes release of completed peptide)

Question 36

terminator tRNA

Answer 36

DOES NOT EXIST

Question 37

Shine-Delgarno sequence

Answer 37

- initiation in prokaryotes - upstream of start codon - directly pairs to rRNA

Question 38

Bacterial ribosomes allow for SELECTIVE inhibition by the ribosome inhibitor class of antibiotics

Answer 38

True

Question 39

Mitochondrial Ribosomes

Answer 39

similar to bacterial ribosomes | - ribosome inhibitors have residual toxicity due to this similarity

Question 40

``` Eukaryote vs Prokaryote Eukaryote - monocistronic mRNA - 40, 60, 80S ribosome - eIF4E - eIF2 - eIF4G - eEF1A - eEF2 ```

Answer 40

Prokaryote - polycistronic - 30, 50, 70S ribosome - eIF4E = no counterpart - eIF2 = IF2 - eIF4G = no counterpart - eEF1A = EFTu - eEF2 = EFG

Question 41

Hypoxia Regulation

Answer 41

- mTOR signaling pathway represses translation in response to hypoxia by regulating the function of eIF4E - the mTOR pathway up-regulates during growth and down-regulates during stress

Question 42

4EBPs

Answer 42

- increased affinity for eIF4E; binds and inhibits translation - when phosphorylated they are released from eIF4E and translation inhibition is relieved

Question 43

4EBP phosphorylation regulator

Answer 43

mTOR pathway; activation causes 4EBP phosphorylation and increases translation

Question 44

mTOR repression mTOR "normal" mTOR high

Answer 44

repression: hypoxia; dephosphorylated, decreased translation normal: normal phosphorylation, normal translation high mTOR: response to growth stimulus or uncontrolled growth (cancer)

Question 45

21st AA

Answer 45

Selenocysteine - AUG codon (NOT stop); codon "re-coded" by SBP2 - cysteine with selenol instead of thiol

Question 46

Hyperthyroidism

Answer 46

caused by selenocysteine deficiency

Question 47

Diptheria toxin

Answer 47

shuts down translation by modifying the elongation factor responsible for translocation (eEF2)

Question 48

Deiodinases

Answer 48

selenoproteins required for thyroid hormone maturation | DIO1-2-3

Question 49

SBP2

Answer 49

protein required for recoding UGA to selenocyteine codon

Question 50

EF1A and selenocysteine

Answer 50

EF1A delivers all tRNA to A site EXCEPT selenocysteine | -eEFSec only protein which can deliver selenocysteine to A site

Question 51

SECIS

Answer 51

HOW mRNA KNOWS IF UGA IS STOP OR SELENOCYSTEINE - when present UGA=> selenocysteine - SBP2 binds exclusively to selenocysteine