Lecture 38: Protein Synthesis I

Question 1

Components of protein synthesis process

Answer 1

1. mRNA
2. Ribosomes (organelle)
3. tRNA (adaptor)
4. Genetic code (language)

Question 2

Features of genetic code in protein synthesis

Answer 2

1. Degenerate (more than 1 codon for some AAs)
2. Not ambiguous (no shared codons)
3. Almost universal (besides mitochondria)

Question 3

Mutation types

Answer 3

1. Point
2. Silent
3. Missense
4. Nonsense
5. Insertion/deletion (frameshift)

Question 4

Point mutation

Answer 4

Single base change

Question 5

Silent mutation

Answer 5

Mutation results in same AA produced (codon degeneracy)

Question 6

Missense mutation

Answer 6

Mutation results in different AA produced

Question 7

Nonsense mutation

Answer 7

Mutation results in new stop codon

Question 8

Insertion/deletion mutation (frameshift)

Answer 8

Adding or deleting 1 or more bases; frameshift if not multiple of 3

Question 9

tRNA anti-codon/codon binding specificity and wobble

Answer 9

5’ and middle base in anti-codon/codon (tRNA/mRNA) interaction must bind perfectly
3’ base has wobble; flexible binding

Question 10

Factor types needed for protein synthesis

Answer 10

1. I (initiation)
2. E (elongation)
3. R (release)

Question 11

AA activation steps

Answer 11

Both steps catalyzed by aminoacyl tRNA synthetase (specific kinds for given AAs)
1. AA + ATP → aminoacyl adenylate:synthetase + PPi
2. Aminoacyl adenylate:synthetase + tRNA → AA-tRNA + AMP + PPi

Question 12

Role of AA activation for protein synthesis

Answer 12

Controls protein synthesis accuracy
1. Each synthetase has to recognize the AA and correct tRNA seq.
2. Each synthetase has activation site + hydrolytic site (error correction)
3. AA-tRNA that leaves cannot be corrected further

Question 13

fMet

Answer 13

Formyl group added to Met w/ initiator tRNA to form fMet; all proteins start w/ fMet

Question 14

Ribosome tRNA-AA sites

Answer 14

1. A site (aminoacyl)
2. P site (peptidyl)
3. E site (exit)

Question 15

Shine-Dalgarno sequence

Answer 15

At 5’ end of prokary. mRNA. Sets reading frame; 1st codon is always AUG (fMet) after Shine-Dalgarno.

Question 16

Prokaryotic translation initiation process

Answer 16

1. Shine-Dalgarno positions 30S subunit
2. Initiator tRNA bound to fMet + IF2-GTP is positioned in P site
3. 50S subunit binds; IF2-GDP + other IFs release
   Thus formation of 70S initiation complex

Question 17

Translation elongation cycle steps

Answer 17

1. EF-Tu-GTP mediates binding codon specific AAcyl-tRNA to A site
2. Peptide bond formation between growing peptide chain/tRNA in A site; mediated by peptidyl transferase ribozyme
3. Free tRNA left in P site
4. Ribosome translocation w/ EF-G-GTP opens new free A site for next tRNA

Question 18

Translation termination process

Answer 18

1. Stop codon appears in A site
2. RF1/2 bind A site
3. RF3 binds common site
4. Peptidyl transferase ribozyme catalyzes ester bond cleavage w/ GTP hydrolysis to release protein, tRNA, mRNA, ribosomal subunits

Question 19

Ribosome common site

Answer 19

EF-TU, EF-G, RF3 all bind same “common” site. This ensures 2 processes can’t occur simultaneously.

Question 20

Polyribosomes

Answer 20

Prokaryotic + eukaryotic feature where many ribosomes translate a gene simultaneously

Question 21

Protein synthesis inhibitors

Answer 21

1. Tetracycline
2. Chloramphenicol
3. Puromycin

Question 22

Tetracycline

Answer 22

Blocks AAcyl-tRNA binding w/ A site in prokaryotes

Question 23

Chloramphenicol

Answer 23

Resembles peptide bond, inhibiting peptidyl transferase in prokaryotes

Question 24

Puromycin

Answer 24

Enters A site and accepts polypeptide chain, blocking translocation of ribosome in prokary. + eukaryotes.

Question 25

Energy use for 100 AA peptide

Answer 25

• AA activation cleaves 2 high energy bonds
• Initiation already places first tRNA in P site
• Subsequent tRNAs need energy for A site binding + translocation (2 bonds)
• Termination uses 1 bond

Question 26

Hep C virus

Answer 26

Flavivirus; HCV virus + strand RNA use host ribosomes to synthesize viral protein. Does NOT insert into DNA

Question 27

HIV

Answer 27

Inserts into genetic material and uses host genetic material to replicate

Question 28

CF mutations

Answer 28

E.g. early stop codon (W1282X) causes premature stop