Protein Synthesis

Question 1

energy demand of protein synthesis

Answer 1

90% of a cell’s energy goes towards protein synthesis
only as many proteins as needed are produced

Question 2

biomolecules required for protein synthesis in eukaryotes

Answer 2

70 ribosomal proteins
20 AA activation enzymes
20 factors for initiation, elongation and termination of translation
100 enzymes for processing
40 kinds of tRNA and rRNA

Question 3

tRNA structure and numbers

Answer 3

cloverleaf shape
- 3’ top arm end is amino acid arm (AA binding site) with CCA residue added post transcription of tRNA
- guanylate residue on 5’ end of top arm
- T-Psi-C - interacts with large subunit rRNA
- anti-codon bottom arm - 5’ wobble position
- D arm - unusual nucleotides here

32 tRNA used to code for 61 codons (31 AA, 1 initiation)

Question 4

genetic code for proteins

Answer 4

3 nucleotide sequences called codons
64 (4^3) total combinations possible (61 AA coding, 3 stop)
successive, non-overlapping code
5’–>3’ reading direction
3rd base is less important in binding to tRNA

Question 5

missense vs nonsense mutation

Answer 5

missense is when the reading frame is off
nonsense is an early stop codon

Question 6

AUG

Answer 6

initiation codon
methionine codon when in internal part of sequence

Question 7

stop codons

Answer 7

UAA, UGA and UAG

Question 8

AA with only 1 codon

Answer 8

Tryptophan is UGG
Methionine is AUG
all AA have a third codon with 2 options, or more

Question 9

mitochondrial different in codon code

Answer 9

Tryptophan is UGA instead of stop
Stop is AGA/AGG instead of Arginine
otherwise prokaryotes and eukaryotes use the same code

Question 10

Wobble position

Answer 10

when I (inosinate) is in anti-codon position 1 (which pairs with codon position 3) can form weak H bonds with A, U or C
gives flexibility for different codons to be recognized by 1 tRNA molecule

Question 11

specific anticodon binding when

Answer 11

Anti-codon position 1 is C or A
Only 1 codon recognized by that tRNA

Question 12

less specific anticodon binding when

Answer 12

1st base of anti-codon is U or G (binding is less specific)
2 different codons can be read

Question 13

Protein synthesis mechanism

Answer 13

occurs in cytosol
1. aminoacylation of tRNA, now charged tRNA
2. Initiation: mRNA and charged tRNA bind to small ribosomal subunit, followed by the large subunit
3. Elongation: successive charged tRNA binding and peptide bond formation as ribosome moves along mRNA until stop codon is reached
4. everything dissociates and ribosomal subunits are recycled (tRNA not recycled)
5. post-translational processing

Question 14

Mechanism of Aminoacylation of tRNA

Answer 14

1. Carboxyl end amino acid attacks alpha phosphate of ATP –> 5’-aminoacyl adenylate (amino acid + AMP)
2. class I: 2-step processing, hydroxyl attack from 2’ OH on ribose of tRNA adenine to carboxyl of 5’amino-acyl adenylate, AMP released, then 3’ OH of ribose attacks C=O in a transesterification

class II: 1 step process, hydroxyl attack of 3’ OH, AMP released and aminoacylated product is produced

Question 15

Aminoacyl-tRNA synthetases

Answer 15

these enzymes aminoacylate the 3’ adenine of tRNA arm
function: to esterify all 20 AA to their right tRNA
each enzyme is specific to 1 AA and 1 or more corresponding tRNA

Question 16

initiation AA in bacteria and mitochondrial mRNA

Answer 16

in bacteria/mitochondria: initiation tRNA inserts N-formylmethionine (fMet), tRNA^fMet
interior bacteria/mitochondria insertion of Met by normal tRNA

In eukaryotes the initiation AA is just Met

Question 17

3 sites that bind tRNA

Answer 17

A - aminoacyl site
P - peptide site
E - exit site

Question 18

Initiation of translation in eukaryotes mechanism

Answer 18

1. Initiation factor bind A site of 40S subunit (small ribosomal) to block premature tRNA binding, along with 2 other factors in E site and on subunit
2. Form 43S Pre-initiation Complex: tRNA binds GTP-bound initiation factor and with another GTP-bound initiation factor binds to 40S subunit
3. mRNA binds mediating factor and associates with 43S to form 48S complex
4. mRNA is scanned from 5’ cap until AUG is found
5. 60S large subunit associates along with release of initiation factors, which completes formation of initiation complex

Question 19

1st peptide bond formation mechanism (prokaryotes)

Answer 19

1. Elongation factor EF-Tu-GTP brings aminoacyl-tRNA to A site
2. EF-Tu-GDP exits, peptide bond is formed between 2 AA
3. EF-G-GTP pushes dipeptidyl tRNA to P site
4. EF-G-GDP exits and A site is empty for another aminoacyl-tRNA

Question 20

termination of protein synthesis mechanism

Answer 20

1. once stop codon is in A site, release factor (RF) binds A site
2. hydrolysis of peptide-tRNA bond in P site by RF followed by release of uncharged tRNA and free polypeptide from P site
3. RF is released
4. RRF (ribosomal recycle factor) and EF-G are replaced by IF3 on 40S subunit which results in tRNA dissociating and large subunit dissociating
5. 40S is now ready to start translation again and mRNA is released

Question 21

post-translational modification of proteins

Answer 21

• enzymatic removal of formyl group
• Met removal or other residues
• acetylation of N-terminal residue
• removal of signal sequences or other regions
• forming disulphide links
• carbohydrate attachment

Question 22

Protein half-lives

Answer 22

ranges from seconds to months
Hemoglobin can live up to 3 months
proteins for rapidly changing needs are short-lived
defective proteins are short-lived
terminal residue indicates half-life

Question 23

E enzymes for Ubiquitination and mechanism

Answer 23

Ubiquitin activating enzymes:
E1 - activating
E2 - conjugating
E3 - ligating
1. Ubiquitin free carboxyl group is bound to E1 (ATP used)
2. Ubiquitin transferred to E2
3. E3 catalyzes transfer of Ubiquitin to target protein

ubiquitin is a regulatory protein

Question 24

how many tRNA are there for 20 AA?

Answer 24

32 tRNA

Question 25

eIF is

Answer 25

eukaryotic initiation factor in translation