Translation

Question 1

Eukaryotic ribosome components

Answer 1

rRNA and protein
60S and 40S subunits
28S rRNA

Question 2

40S subunit

Answer 2

Scanning subunit of ribosome: looks for start codon

Question 3

28S rRNA

Answer 3

Part of ribosome that catalyzes peptide bond formation (peptidyl transferase)

Question 4

What makes the ribosome a ribozyme?

Answer 4

RNA portion catalyzes peptide bond formation

No amino acids are present in the ribosome’s active site

Question 5

Ribosome cycle

Answer 5

2 subunits of ribosome associate
Ribosome translates starting at methionine (AUG) and continues in 5’ -> 3’ direction
Once the ribosome reaches the stop codon, it dissociates and can go off and translate another mRNA

Question 6

Polysome

Answer 6

Multiple ribosomes/mRNA

Question 7

tRNA structure

Answer 7

Cloverleaf structure (in real life, looks like an L)
Going clockwise from top: acceptor arm, pseudo U loop (modified uracils), variable loop, anticodon loop, D loop (dihydrouridines)

Question 8

Acceptor arm

Answer 8

Portion of tRNA where amino acid is added

Has a CCA sequence on its 3’ end (not part of original tRNA; added by CCA adding enzyme)

Question 9

Anticodon loop

Answer 9

Portion of tRNA that binds to mRNA that is being translated

Question 10

tRNA charging steps

Answer 10

1. -O of -CO2- of amino acid attacks alpha -PO3 of ATP, forming adenylylated amino acid
2. -OH at terminal adenine of tRNA attaches to the ester C atom of the adenylylated amino acid (covalent addition of tRNA to amino acid through 3’ adenine)
3. High energy bond is created for use in peptide bond formation (tRNA is charged)

Question 11

How ribosome controls peptide bond formation

Answer 11

Ribosome goes by complementary base pairing between mRNA codon and tRNA anticodon
If the tRNA is incorrectly charged, then the wrong amino acid is added

Question 12

Which component of the translation machinery is capable of proofreading and editing amino acids?

Answer 12

tRNA synthetase, which recognizes features of the amino acids’ R-groups
Certain amino acids can’t bind (synthetase for phenylalanine blocks out -OH group of tyrosine; isoleucine fits more easily than valine in isoleucyl-tRNA synthetase)

Question 13

Coupling of transcription and translation

Answer 13

Transcription and translation are coupled in prokaryotes, but not in eukaryotes

Question 14

Sequences involved in initiation of translation in prokaryotes vs. eukaryotes

Answer 14

Prokaryotes: Shine-Dalgarno sequence
Eukaryotes: Kozak sequence

Question 15

Starting tRNA in prokaryotes vs. eurkaryotes

Answer 15

Prokaryotes: fMet-tRNA^fMet
Eukaryotes: Met-tRNA^Met

Question 16

Translation initiation steps

Answer 16

1. Initiation factors keep large/small ribosome subunits apart
2. eIF2 (eukaryotic initiation factor 2) bound to GTP brings in Met-tRNAi^Met
3. eIF4E binds 5’ cap of mRNA, and eIF4G and eIF4A bind as well
4. eIF4A unwinds secondary structure of mRNA using helicase activity
5. eIF4B binds to the mRNA
6. mRNA and eIF4E, G, A, and B bind to 43S complex, forming the 48S pre-initiation complex

Question 17

Steps in translation initiation through scanning

Answer 17

1. 48S moves along the 5’ UTR (untranslated region)
2. Base pairing occurs between AUG start and Met-tRNAi^Met anticodon
3. IFs are released
4. Large ribosomal subunit can bind to the small subunit
5. eIF5B hydrolyzes GTP
6. 80S complex can start translating

Question 18

Circular translation

Answer 18

mRNA forms a circle: eIF4G is bound to the poly A tail and to eIF4E, which is bound to the cap

Question 19

Steps in translation

Answer 19

1. Aminoacyl tRNA enters A site
2. Peptide bond formation occurs between incoming amino acid and polypeptide
3. Ribosome moves down 1 codon (translocation)

Question 20

3 sites of ribosome

Answer 20

A site (aminoacyl site; entry site of tRNA)
P site (peptidyl site; where tRNA with polypeptide chain waits for new tRNA)
E site (exit site; from where empty tRNA leaves)

Question 21

Peptidyl transferase reaction

Answer 21

-NH2 of amino acid of aminoacyl-tRNA attacks primary -COO of peptidyl-tRNA
Aminoacyl-tRNA gains polypeptide chain, and peptidyl-tRNA is left with just an -OH group

Question 22

Elongation factor Tu (EF-Tu)

Answer 22

Escorts tRNA to the A site of the ribosome (binds to top of tRNA)
EF-Tu-GTP fits into the factor binding center (above the A site)
GTP is hydrolyzed by EF-Tu
EF-Tu changes shape (new shape has low affinity for the factor binding center) and dissociates

Question 23

A site specificity

Answer 23

H-bonding between anticodon, mRNA, and 16S rRNA doesn’t occur if anticodon of tRNA is incorrect
Accomodation: tRNA turns in the A site if amino acid is correct (if amino acid isn’t correct, tRNA doesn’t turn and is ejected from ribosome)
If anticodon of tRNA is incorrect, then EF-Tu can’t fit into the factor binding center and dissociates

Question 24

Translocation steps

Answer 24

Polypeptide chain is transferred to tRNA in A site, making a hybrid state which is unfavorable

1. EF-G-GTP fits into the factor binding site
2. EF-G moves the ribosome (or mRNA; not totally sure which) over 1 codon, moving the tRNA in the A site to the P site
3. EF-G-GTP and tRNA in E site dissociate

Question 25

Polypeptide release steps

Answer 25

1. Release factor 1 (RF1) peptide anticodon interacts with the stop codon 2. Polypeptide is released 3. RF3-GDP comes in and interacts with RF1 4. RF3 exchanges GDP for GTP and releases RF1 5. RF3-GDP dissociates from the complex

Question 26

Ribosome recycling steps

Answer 26

1. Ribosome recycling factor (RRF) binds to the A site 2. EF-G-GTP also binds to the A site 3. GTP is hydrolyzed to GDP 4. New shapes of RRF and EF-G cause dissociation of complex 5. IF3 binds to keep the large and small subunits apart

Question 27

Ways to regulate translation in eukaryotes

Answer 27

1. Inhibit proteins that are involved in initiation 2. Upstream ORFs (if translated, main gene isn't translated) 3. Nonsense-mediated decay

Question 28

Nonsense-mediated decay

Answer 28

Occurs during pioneer round of translation Ribosome pauses at pre-mature stop codon and signals that something is wrong Upf proteins bring in de-capping and de-adenylating enzymes (degradation of mRNA occurs from both directions)